ATM inhibitors

ABSTRACT

The application concerns a compound of formula I:  
                 
 
     wherein one of P and Q is O, and the other of P and Q is CH, where there is a double bond between whichever of Q and P is CH and the carbon atom bearing the R 3  group;  
     Y is either O or S;  
     R 1  and R 2  are independently hydrogen, an optionally substituted C 1-7  alkyl group, C 3-20  heterocyclyl group, or C 5-20  aryl group, or may together form an optionally substituted heterocyclic ring having from 4 to 8 ring atoms;  
     R 3  is a phenyl or pyridyl group, attached by a first bridge group selected from —S—, —S(═O)—, —S(═O) 2 —, —O—, —NR N — and CR C1 R C2 — to an optionally substituted C 5-20  carboaryl group, the phenyl or pyridyl group and optionally substituted C 5-20  carboaryl group being optionally further linked by a second bridge group, so as to form an optionally substituted C 5-7  ring, the phenyl or pyridyl group being further optionally substituted.

[0001] The present invention relates to compounds which act as ATMinhibitors, their use and synthesis.

[0002] Human DNA is constantly under attack from,reactive oxygenintermediates principally from by-products of oxidative metabolism.Reactive oxygen species are capable of producing DNA single-strandbreaks and, where two of these are generated in close proximity, DNAdouble strand breaks (DSBs). In addition, single- and double-strandbreaks can be induced when a DNA replication fork encounters a damagedtemplate, and are generated by exogenous agents such as ionisingradiation (IR) and certain anti-cancer drugs (e.g. bleomycin, etoposide,camptothecin). DSBs also occur as intermediates in site-specific V(D)Jrecombination, a process that is critical for the generation of afunctional vertebrate immune system. If DNA DSBs are left unrepaired orare repaired inaccurately, mutations and/or chromosomal aberrations areinduced, which in turn may lead to cell death. To combat the seriousthreats posed by DNA DSBs, eukaryotic cells have evolved severalmechanisms to mediate their repair. Critical to the process of DNArepair is the slowing down of cellular proliferation to allow time forthe cell to repair the damage. A key protein in the detection of DNADSBs and in the signalling of this information to the cell cyclemachinery is the kinase ATM (ataxia telangiectasia mutated) (Durocherand Jackson (2001) DNA-PK, ATM and ATR as sensors of DNA damage:variations on a theme? Curr Opin Cell Biol., 13:225-31, Abraham (2001)Cell cycle checkpoint signaling through the ATM and ATR kinases. GenesDev., 15; 2177-96).

[0003] The ATM protein is an ˜350 kDa polypeptide that is a member ofthe phosphatidylinositol (PI) 3-kinase family of proteins by virtue of aputative kinase domain in its carboxyl-terminal region (Savitsky et al(1995) A single ataxia telangiectasia gene with a product similar toPI-3 kinase. Science, 268:1749-53). Classical PI 3-kinases, such as PI3-kinase itself, are involved in signal transduction and phosphorylateinositol lipids that act as intracellular second messengers (reviewed inToker and Cantley (1997) Signalling through the lipid products ofphosphoinositide-3-OH kinase, Nature, 387: 673-6). However, ATM bearsmost sequence similarity with a subset of the PI 3-kinase family thatcomprises proteins which, like ATM, are involved in cell cycle controland/or in the detection and signalling of DNA damage (Keith andSchreiber (1995) PIK-related kinases: DNA repair, recombination, andcell cycle checkpoints, Science, 270; 50-1, Zakian (1995) ATM-relatedgenes: what do they tell us about functions of the human gene? Cell, 82;685-7). Notably there is no evidence to date that any members of thissubset of the PI 3-kinase family are able to phosphorylate lipids.However, all members of this family have been shown to possessserine/threonine kinase activity. ATM phosphorylates key proteinsinvolved in a variety of cell-cycle checkpoint signalling pathways thatare initiated in response to DNA DSBs production (see below). Thesedownstream effector proteins include p53, Chk2, NBS1/nibrin, BRCA1 andRad 17 (Abraham, 2001)

[0004] ATM is the product of the gene mutated in ataxia-telangiectasia(A-T) (Savitsky et al (1995)). A-T is a human autosomal recessivedisorder present at an incidence of around 1 in 100,000 in thepopulation. A-T is characterised by a number of debilitating symptoms,including progressive cerebellar degeneration, occulocutaneoustelangiectasia, growth retardation, immune deficiencies, cancerpredisposition and certain characteristics of premature ageing (Lavinand Shiloh (1997), The genetic defect in ataxia-telangiectasia. Annu.Rev. Immunol., 15:177-202; Shiloh (2001), ATM and ATR: networkingcellular responses to DNA damage, Curr. Opin. Genet. Dev., 11:71-7). Atthe cellular level, A-T is characterised by a high degree of chromosomalinstability, radio-resistant DNA synthesis, and hypersensitivity toionizing radiation (IR) and radiomimetic drugs. In addition, A-T cellsare defective in the radiation induced G₁-S, S, and G₂-M cell cyclecheckpoints that are thought to arrest the cell cycle in response to DNAdamage in order to allow repair of the genome prior to DNA replicationor mitosis (Lavin and Shiloh, 1997). This may in part reflect the factthat A-T cells exhibit deficient or severely delayed induction of p53 inresponse to IR. Indeed, p53-mediated downstream events are alsodefective in A-T cells following IR exposure. ATM therefore actsupstream of p53 in an IR-induced DNA damage signalling pathway. A-Tcells have also been shown to accumulate DNA double-strand breaks (dsbs)after ionizing radiation, suggesting a defect in dsb repair.

[0005] It is clear that ATM is a key regulator of the cellular responseto DNA DSBs. Therefore the inhibition of this kinase through smallmolecules will sensitise cells to both ionising radiation and tochemotherapeutics that induce DNA DSBs either directly or indirectly.ATM inhibitors may thus be used as adjuncts in cancer radiotherapy andchemotherapy. To date the only reported inhibitors of ATM (caffeine andwortmannin; Sarkaria, et al., (1999) Inhibition of ATM and ATR kinaseactivities by the radiosensitizing agent, caffeine. Cancer Res.,59:4375-82; Banin, et al., (1998) Enhanced phosphorylation of p53 by ATMin response to DNA damage.

[0006]Science, 281:1674-1677) do cause radiosensitisation but it isunclear whether this mechanism of action is mediated through ATMinhibition as these small molecules are very non-specific in action askinase inhibitors.

[0007] ATM function in response to ionising radiation induced DNA damagehas been shown to be tissue specific. For example, while fibroblastsderived from Atm null mice are radiosensitive Atm null neurons areradioresistant through a lack of IR induced apoptosis (Herzog et al.,(1998) Requirement for Atm in ionizing radiation-induced cell death inthe developing central nervous system. Science, 280: 1089-91).Therefore, inhibitors of ATM have the potential to be radio-protectivein specific cellular contexts.

[0008] ATM inhibitors may also prove useful in the treatment ofretroviral mediated diseases. It has been demonstrated that ATM functionis required to allow stable retroviral DNA transduction under certainconditions (Daniel et al. (2001) Wortmannin potentiatesintegrase-mediated killing of lymphocytes and reduces the efficiency ofstable transduction by retroviruses. Mol. Cell Biol., 21: 1164-72).Therefore ATM inhibitors have the potential to block retroviral DNAintegration.

[0009] ATM is known to play a crucial role in controlling the length oftelomeric chromosomal ends (Metcalfe et al. (1996) Accelerated telomereshortening in ataxia telangiectasia. Nat Genet., 13 :350-3). Telomericends in most normal cell types shorten at each cell division. Cells withexcessively shortened telomeres are unable to divide. Inhibitors of ATMmay therefore, have utility in preventing cancer progression by limitingthe growth potential of cancerous or pre-cancerous cells. Furthermore,ATM does not appear to be part of the telomerase enzyme itself (Metcalfeet al. (1996)) Therefore it is likely that ATM inhibitors will worksynergistically with anti-telomerase drugs.

[0010] Cells derived from A-T patients or from mice null for ATM growslower in culture than genetically matched ATM positive cells. Thereforean ATM inhibitor may have growth inhibitory/anti-proliferativeproperties in its own right. Therefore an ATM inhibitor may be used as acytostatic agent in the treatment of cancer.

[0011] A-T patients display immuno-deficiencies, demonstrating that ATMis required for generation of a fully functional immune system.Inhibitors of ATM may, therefore, be used in modulating the immunesystem.

[0012] In summary ATM inhibitors have the potential to sensitise tumourcells to ionising radiation or DNA DSB inducing chemotherapeutics, tomodulate telomere length control mechanisms, to block retroviralintegration, modulate the immune system and to protect certain celltypes from DNA damage induced apoptosis.

[0013] The present inventors have now discovered compounds which exhibitinhibition of ATM.

[0014] Accordingly, the first aspect of the invention provides acompound of formula I:

[0015] and isomers, salts, solvates, chemically protected forms, andprodrugs thereof, wherein:

[0016] one of P and Q is O, and the other of P and Q is CH, where thereis a double bond between whichever of Q and P is CH and the carbon atombearing the R³ group;

[0017] Y is either O or S;

[0018] R¹ and R² are independently hydrogen, an optionally substitutedC₁₋₇ alkyl group, C₃₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or maytogether form, along with the nitrogen atom to which they are attached,an optionally substituted heterocyclic ring having from 4 to 8 ringatoms;

[0019] R³ is a phenyl or pyridyl group, attached by a first bridge groupselected from —S—, —S(═O)—, —S(═O)₂—, —O—, —NR^(N)— and CR^(C1)R^(C2)—to an optionally substituted C₅₋₂₀ carboaryl group, in which onearomatic ring atom may be replaced by a nitrogen ring atom;

[0020] the phenyl or pyridyl group and optionally substituted C₅₋₂₀carboaryl group being optionally further linked by a second bridgegroup, which is bound adjacent the first bridge group on both groups soas to form an optionally substituted C₅₋₇ ring fused to both the phenylor pyridyl group and the C₅₋₂₀ carboaryl group, the phenyl or pyridylgroup being further optionally substituted;

[0021] wherein R^(N) is selected from hydrogen, an ester group, anoptionally substituted C₁₋₇ alkyl group, an optionally substituted C₃₋₂₀heterocyclyl group and an optionally substituted C₅₋₂₀ aryl group;

[0022] and R^(C1) and R^(C2) are independently selected from hydrogen,an optionally substituted C₁₋₇ alkyl group, an optionally substitutedC₃₋₂₀ heterocyclyl group and an optionally substituted C₅₋₂₀ aryl group.

[0023] Therefore, when P is O and Q is CH, the compound is of formula(Ia):

[0024] and when P is CH and Q is O, the compound is of formula (Ib):

[0025] A second aspect of the invention provides a compositioncomprising a compound of the first aspect and a pharmaceuticallyacceptable carrier or diluent.

[0026] A third aspect of the invention provides methods of treating acondition in a patient which is known to be ameliorated by theinhibition of ATM comprising administering to said patient atherapeutically-effective amount of a compound of the first aspect.

[0027] A fourth aspect of the invention provides methods of treatingcancer in a patient comprising administering to said patient atherapeutically-effective amount of a compound of the first aspect incombination with ionising radiation or a chemotherapeutic agent.

[0028] A fifth aspect of the invention provides methods of treating aretroviral mediated disease, including acquired immunodeficiencysyndrome, in a patient comprising administering to said patient atherapeutically-effective amount of a compound of the first aspect.

[0029] Another aspect of the invention provides a method of inhibitingATM in vitro or in vivo, comprising contacting a cell with an effectiveamount of a compound of the first aspect.

Definitions

[0030] C₁₋₇ alkyl: The term “C₁₋₇ alkyl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a C₁₋₇hydrocarbon compound having from 1 to 7 carbon atoms, which may bealiphatic or alicyclic, or a combination thereof, and which may besaturated, partially unsaturated, or fully unsaturated.

[0031] Examples of saturated linear C₁₋₇ alkyl groups include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl).

[0032] Examples of saturated branched C₁₋₇ alkyl groups include, but arenot limited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, andneo-pentyl.

[0033] Examples of saturated alicyclic C₁₋₇ alkyl groups (also referredto as “C₃₋₇ cycloalkyl” groups) include, but are not limited to, groupssuch as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well assubstituted groups (e.g., groups which comprise such groups), such asmethylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl,dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl,methylcyclohexyl, dimethylcyclohexyl, cyclopropylmethyl andcyclohexylmethyl.

[0034] Examples of unsaturated C₁₇ alkyl groups which have one or morecarbon-carbon double bonds (also referred to as “C₂₋₇alkenyl” groups)include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 2-propenyl(allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂), butenyl, pentenyl, andhexenyl.

[0035] Examples of unsaturated C₁₋₇ alkyl groups which have one or morecarbon-carbon triple bonds (also referred to as “C₂₋₇ alkynyl” groups)include, but are not limited to, ethynyl (ethinyl) and 2-propynyl(propargyl).

[0036] Examples of unsaturated alicyclic (carbocyclic) C₁₋₇ alkyl groupswhich have one or more carbon-carbon double bonds (also referred to as“C₃₋₇ cycloalkenyl” groups) include, but are not limited to,unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl,and cyclohexenyl, as well as substituted groups (e.g., groups whichcomprise such groups) such as cyclopropenylmethyl andcyclohexenylmethyl.

[0037] C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl”, as usedherein, pertains to a monovalent moiety obtained by removing a hydrogenatom from a ring atom of a C₃₋₂₀ heterocyclic compound, said compoundhaving one ring, or two or more rings (e.g., spiro, fused, bridged), andhaving from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ringheteroatoms, and wherein at least one of said ring(s) is a heterocyclicring.

[0038] Preferably, each ring has from 3 to 7 ring atoms, of which from 1to 4 are ring heteroatoms. “C₃₋₂₀” denotes ring atoms, whether carbonatoms or heteroatoms.

[0039] Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ringatom include, but are not limited to, those derived from aziridine,azetidine, pyrrolidines (tetrahydropyrrole), pyrroline (e.g.,3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (isopyrrole,isoazole), piperidine, dihydropyridine, tetrahydropyridine, and azepine.

[0040] Examples of C₃₋₂₀ heterocyclyl groups having one oxygen ring atominclude, but are not limited to, those derived from oxirane, oxetane,oxolane (tetrahydrofuran), oxole (dihydrofuran), oxane(tetrahydropyran), dihydropyran, pyran (C₆), and oxepin. Examples ofsubstituted C₃₋₂₀ heterocyclyl groups include sugars, in cyclic form,for example, furanoses and pyranoses, including, for example, ribose,lyxose, xylose, galactose, sucrose, fructose, and arabinose.

[0041] Examples of C₃₋₂₀ heterocyclyl groups having one sulphur ringatom include, but are not limited to, those derived from thiirane,thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran),and thiepane.

[0042] Examples of C₃₋₂₀ heterocyclyl groups having two oxygen ringatoms include, but are not limited to, those derived from dioxolane,dioxane, and dioxepane.

[0043] Examples of C₃₋₂₀ heterocyclyl groups having two nitrogen ringatoms include, but are not limited to, those derived from imidazolidine,pyrazolidine (diazolidine), imidazoline, pyrazoline (dihydropyrazole),and piperazine.

[0044] Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ringatom and one oxygen ring atom include, but are not limited to, thosederived from tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole,dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, andoxazine.

[0045] Examples of C₃₋₂₀ heterocyclyl groups having one oxygen ring atomand one sulphur ring atom include, but are not limited to, those derivedfrom oxathiolane and oxathiane (thioxane).

[0046] Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ringatom and one sulphur ring atom include, but are not limited to, thosederived from thiazoline, thiazolidine, and thiomorpholine.

[0047] Other examples of C₃₋₂₀heterocyclyl groups include, but are notlimited to, oxadiazine and oxathiazine.

[0048] Examples of heterocyclyl groups which additionally bear one ormore oxo (═O) groups, include, but are not limited to, those derivedfrom:

[0049] C₅ heterocyclics, such as furanone, pyrone, pyrrolidone(pyrrolidinone), pyrazolone (pyrazolinone), imidazolidone, thiazolone,and isothiazolone;

[0050] C₆heterocyclics, such as piperidinone (piperidone),piperidinedione, piperazinone, piperazinedione, pyridazinone, andpyrimidinone (e.g., cytosine, thymine, uracil), and barbituric acid;

[0051] fused heterocyclics, such as oxindole, purinone (e.g., guanine),benzoxazolinone, benzopyrone (e.g., coumarin);

[0052] cyclic anhydrides (—C(═O)—O—C(═O)— in a ring), including but notlimited to maleic anhydride, succinic anhydride, and glutaric anhydride;

[0053] cyclic carbonates (—O—C(═O)—O— in a ring), such as ethylenecarbonate and 1,2-propylene carbonate;

[0054] imides (—C(═O)—NR—C(═O)— in a ring), including but not limitedto, succinimide, maleimide, phthalimide, and glutarimide;

[0055] lactones (cyclic esters, —O—C(═O)— in a ring), including, but notlimited to, β-propiolactone, γ-butyrolactone, δ-valerolactone(2-piperidone), and ε-caprolactone;

[0056] lactams (cyclic amides, —NR—C(═O)— in a ring), including, but notlimited to, β-propiolactam, γ-butyrolactam (2-pyrrolidone),δ-valerolactam, and ε-caprolactam;

[0057] cyclic carbamates (—O—C(═O)—NR— in a ring), such as2-oxazolidone;

[0058] cyclic ureas (—NR—C(═O)—NR— in a ring), such as 2-imidazolidoneand pyrimidine-2,4-dione (e.g., thymine, uracil).

[0059] C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of a C₅₋₂₀ aromatic compound, said compound having one ring,or two or more rings (e.g. fused), and having from 5 to 20 ring atoms,and wherein at least one of said ring(s) is an aromatic ring.Preferably, each ring has from 5 to 7 ring atoms.

[0060] The ring atoms may be all carbon atoms, as in “carboaryl groups”,in which case the group may conveniently be referred to as a “C₅₋₂₀carboaryl” group.

[0061] Examples of C₅₋₂₀ aryl groups which do not have ring heteroatoms(i.e. C₅₋₂₀ carboaryl groups) include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), anthracene(C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

[0062] Examples of aryl groups which comprise fused rings, one of whichis not an aromatic ring, include, but are not limited to, groups derivedfrom indene and fluorene.

[0063] Alternatively, the ring atoms may include one or moreheteroatoms, including but not limited to oxygen, nitrogen, and sulphur,as in “heteroaryl groups”. In this case, the group may conveniently bereferred to as a “C₅₋₂₀ heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

[0064] Examples of C₅₋₂₀ heteroaryl groups include, but are not limitedto, C₅ heteroaryl groups derived from furan (oxole), thiophene (thiole),pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole),triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, andoxatriazole; and C₆ heteroaryl groups derived from isoxazine, pyridine(azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole,and oxadiazole (furazan).

[0065] Examples of C₅₋₂₀ heteroaryl groups which comprise fused rings,include, but are not limited to, C₉ heterocyclic groups derived frombenzofuran, isobenzofuran, indole, isoindole, purine (e.g., adenine,guanine), benzothiophene, benzimidazole; C₁₀ heterocyclic groups derivedfrom quinoline, isoquinoline, benzodiazine, pyridopyridine, quinoxaline;C₁₃ heterocyclic groups derived from carbazole, dibenzothiophene,dibenzofuran; C₁₄ heterocyclic groups derived from acridine, xanthene,phenoxathiin, phenazine, phenoxazine, phenothiazine.

[0066] The above C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl, and C₅₋₂₀ aryl groups,whether alone or part of another substituent, may themselves optionallybe substituted with one or more groups selected from themselves and theadditional substituents listed below.

[0067] Halo: —F, —Cl, —Br, and —I.

[0068] Hydroxy: —OH.

[0069] Ether: —OR, wherein R is an ether substituent, for example, aC₁₋₇ alkyl group (also referred to as a C₁₋₇ alkoxy group, discussedbelow), a C₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀heterocyclyloxy group), or a C₅₋₂₀ aryl group (also referred to as aC₅₋₂₀ aryloxy group), preferably a C₁₋₇ alkyl group.

[0070] C₁₋₇ alkoxy: —OR, wherein R is a C₁₋₇ alkyl group. Examples ofC₁₋₇ alkoxy groups include, but are not limited to, —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy) and —OC(CH₃)₃ (tert-butoxy).

[0071] C₁₋₂ alkdioxylene: The term “C₁₋₂ alkdioxylene,” as used herein,pertains to a bidentate moiety obtained by removing two hydrogen atomsfrom each of two different alcohol groups of a C₁₋₂ hydrocarbon diolcompound having from 1 or 2 carbon atoms, i.e. CH₂(OH)₂ andHO—CH₂—CH₂—OH, to form —O—CH₂—O— and —O—CH₂—CH₂—O—. This bidentatemoiety may be the substituent group of a single atom or of two adjacentatoms.

[0072] Oxo (keto, -one): ═O. Examples of cyclic compounds and/or groupshaving, as a substituent, an oxo group (═O) include, but are not limitedto, carbocyclics such as cyclopentanone and cyclohexanone;heterocyclics, such as pyrone, pyrrolidone, pyrazolone, pyrazolinone,piperidone, piperidinedione, piperazinedione, and imidazolidone; cyclicanhydrides, including but not limited to maleic anhydride and succinicanhydride; cyclic carbonates, such as propylene carbonate; imides,including but not limited to, succinimide and maleimide; lactones(cyclic esters, —O—C(═O)— in a ring), including, but not limited to,β-propiolactone, γ-butyrolactone, δ-valerolactone, and ε-caprolactone;and lactams (cyclic amides, —NH—C(═O)— in a ring), including, but notlimited to, β-propiolactam, γ-butyrolactam (2-pyrrolidone),δ-valerolactam, and ε-caprolactam.

[0073] Imino (imine): ═NR, wherein R is an imino substituent, forexample, hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples ofester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

[0074] Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

[0075] Acyl (keto): —C(═O)R, wherein R is an acyl substituent, forexample, a C₁₋₇alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇alkanoyl), a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀heterocyclylacyl), or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀arylacyl), preferably a C₁₋₇ alkyl group. Examples of acyl groupsinclude, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃(propionyl), —C(═O)C(CH₃)₃ (butyryl), and —C(═O)Ph (benzoyl, phenone).

[0076] Carboxy (carboxylic acid): —COOH.

[0077] Ester (carboxylate, carboxylic acid ester, oxycarbonyl):—C(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇alkyl-group. Examples of ester groups include, but are not limitedto, —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

[0078] Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxysubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples ofacyloxy groups include, but are not limited to, —OC(═O)CH₃ (acetoxy),—OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

[0079] Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide):—C(═O)NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of amido groups include, but are notlimited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

[0080] Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amidesubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group, and R² is an acyl substituent, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferablyhydrogen or a C₁₋₇ alkyl group. Examples of acylamide groups include,but are not limited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹and R² may together form a cyclic structure, as in, for example,succinimidyl, maleimidyl and phthalimidyl:

[0081] Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

[0082] Tetrazolyl: a five membered aromatic ring having four nitrogenatoms and one carbon atom,

[0083] Amino: —NR¹R², wherein R¹ and R² are independently aminosubstituents, for example, hydrogen, a C₁₋₇ alkyl group (also referredto as C₁₋₇ alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇alkyl group, or, inthe case of a “cyclic” amino group, R¹ and R², taken together with thenitrogen atom to which they are attached, form a heterocyclic ringhaving from 4 to 8 ring atoms. Examples of amino groups include, but arenot limited to, —NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and—NHPh. Examples of cyclic amino groups include, but are not limited to,aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino,and thiomorpholino.

[0084] Imino: ═NR, wherein R is an imino substituent, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇ alkyl group.

[0085] Amidine: —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. An example ofan amidine group is —C(═NH)NH₂.

[0086] Nitro: —NO₂.

[0087] Nitroso: —NO.

[0088] Azido: —N₃.

[0089] Cyano (nitrile, carbonitrile): —CN.

[0090] Isocyano: —NC.

[0091] Cyanato: —OCN.

[0092] Isocyanato: —NCO.

[0093] Thiocyano (thiocyanato): —SCN.

[0094] Isothiocyano (isothiocyanato): —NCS.

[0095] Sulfhydryl (thiol, mercapto): —SH.

[0096] Thioether (sulfide): —SR, wherein R is a thioether substituent,for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthiogroup), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

[0097] Disulfide: —SS—R, wherein R is a disulfide substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇alkyl disulfide). Examples of C₁₋₇ alkyl disulfide groups include, butare not limited to, —SSCH₃ and —SSCH₂CH₃.

[0098] Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfone groupsinclude, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl),—S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃, —S(═O)₂C₄F₉ (nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂Ph (phenylsulfonyl), 4-methylphenylsulfonyl(tosyl), 4-bromophenylsulfonyl (brosyl), and 4-nitrophenyl (nosyl).

[0099] Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

[0100] Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxygroups include, but are not limited to, —OS(═O)₂CH₃ and —OS(═O)₂CH₂CH₃.

[0101] Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxygroups include, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

[0102] Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, asdefined for amino groups. Examples of sulfamino groups include, but arenot limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

[0103] Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, asdefined for amino groups, and R is a sulfonamino substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonamino groupsinclude, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

[0104] Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, asdefined for amino groups, and R is a sulfinamino substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfinamino groupsinclude, but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

[0105] Sulfamyl: —S(═O)NR¹R², wherein R¹ and R² are independently aminosubstituents, as defined for amino groups. Examples of sulfamyl groupsinclude, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃),—S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

[0106] Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, asdefined for amino groups, and R is a sulfonamino substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonamino groupsinclude, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅. Aspecial class of sulfonamino groups are those derived from sultams—inthese groups one of R¹ and R is a C₅₋₂₀ aryl group, preferably phenyl,whilst the other of R¹ and R is a bidentate group which links to theC₅₋₂₀ aryl group, such as a bidentate group derived from a C₁₋₇ alkylgroup. Examples of such groups include, but are not limited to:

[0107] 2,3-dihydro-tenzo[d]isothiazole-1,1-dioxide-2-yl

[0108] 1,3-dihydro-benzo[c]isothiazole-2,2-dioxide-1-yl

[0109] 3,4-dihydro-2H-benzo[e][1,2]thiazine-1,1-dioxide-2-yl

[0110] Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² arephosphoramidite substituents, for example, —H, a (optionallysubstituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphoramidite groups include, but are not limited to,—OP(OCH₂CH₃)—N(CH₃)₂, —OP(OCH₂CH₃)—N(i-Pr)₂, and—OP(OCH₂CH₂CN)—N(i-Pr)₂.

[0111] Phosphoramidate: —OP(═O) (OR¹)—NR² ₂, where R¹ and R² arephosphoramidate substituents, for example, —H, a (optionallysubstituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphoramidate groups include, but are not limited to,—OP(═O) (OCH₂CH₃)—N(CH₃)₂, —OP(═O) (OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

[0112] In many cases, substituents may themselves be substituted. Forexample, a C₁₋₇ alkoxy group may be substituted with, for example, aC₁₋₇ alkyl (also referred to as a C₁₋₇ alkyl—C₁₋₇alkoxy group), forexample, cyclohexylmethoxy, a C₃₋₂₀ heterocyclyl group (also referred toas a C₅₋₂₀ aryl—C₁₋₇ alkoxy group), for example phthalimidoethoxy, or aC₅₋₂₀ aryl group (also referred to as a C₅₋₂₀aryl-C₁₋₇alkoxy group), forexample, benzyloxy.

C₅₋₇ Ring

[0113] The C₅₋₇ ring in R³ has at least two carbon-carbon double bond,by virtue of its fusion to a benzene or pyridine ring and a C₅₋₂₀carboaryl group. If the C₅₋₂₀ carboaryl group contains a nitrogen ringatom, this does not form part of the C₅₋₇ ring. The same applies to thenitrogen ring atom of the pyridyl group.

[0114] Thus, the C₅₋₇ ring may be a C₅₋₇ sulphur containing heterocycle,a C₅₋₇ oxygen heterocycle, a C₅₋₇ nitrogen containing heterocycle or aC₅₋₇ cyclic group containing at least 5 carbon ring atoms.

[0115] The second bridge group may typically be a single bond (resultingin a C₅ ring), or have 1 or 2 atoms in a chain (resulting in C₆ and C₇rings respectively), which atoms are usually selected from C, S, O andN, with substitution as appropriate.

C₅₋₇ Sulphur Containing Heterocycle

[0116] The C₅₋₇ sulphur containing heterocycle in R³ will have at leasttwo carbon-carbon double bonds, by virtue of its fusion to a benzene orpyridine ring and a C₅₋₂₀ carboaryl group. Examples of relevant C₅₋₇sulphur containing heterocycles include, but are not limited to:

[0117] The C₅₋₇ sulphur containing heterocycle may be substituted (whenpossible) by the,substituent group listed above.

[0118] The groups shown above may in particular be substituted on thesulphur atom in the first bridge group by one or two oxo (═O) groups.

C₅₋₇ Oxygen Containing Heterocycle

[0119] The C₅₋₇ oxygen containing heterocycle in R³ will have at leasttwo carbon-carbon double bonds, by virtue of its fusion to a benzene orpyridine ring and a C₅₋₂₀ carboaryl group. Examples of relevant C₅₋₇oxygen containing heterocycles include, but are not limited to:

[0120] The C₅₋₇ oxygen containing heterocycle may be substituted (whenpossible) by the substituent groups listed above.

C₅₋₇ Nitrogen Containing Heterocycle

[0121] The C₅₋₇ nitrogen containing heterocycle in R³ will have at leasttwo carbon-carbon double bonds, by virtue of its fusion to a benzene orpyridine ring and a C₅₋₂₀ carboaryl group. Examples of relevant C₅₋₇nitrogen containing heterocycles include, but are not limited to(illustrated with R^(N)═H):

[0122] The C₅₋₇ nitrogen containing heterocycle may be substituted (whenpossible) by the substituent group listed above. In particular, thenitrogen atom in the first bridge group may be substituted by R^(N).

C₅₋₇ Cyclic Group Containing at Least 5 Carbon Ring Atoms

[0123] The C₅₋₇ cyclic group containing at least 5 carbon ring atoms inR³ will have at least two carbon-carbon double bonds, by virtue of beingfused to a benzene or pyridine ring and a C₅₋₂₀ carboaryl group.Examples of relevant C₅₋₇ cyclic group containing at least 5 carbon ringatoms include, but are not limited to:

[0124] The C₅₋₇ cyclic group containing at least 5 carbon ring atoms maybe substituted (when possible) by the substituent group listed above.

Possible R³ Structures

[0125] Accordingly, when the phenyl or pyridyl group is linked to aC₅₋₂₀ carboaryl group, R³ can be of the following structure, wherein thephenyl or pyridyl group and the C₅₋₂₀ carboaryl group are illustrated asbenzene rings, without being limited thereto, and where X may be O, S,S(═O), S(═O)₂, NR^(N) and CR^(C1)R^(C2):

[0126] with substitution as appropriate on the above core structures

[0127] When the phenyl or pyridyl group is linked to a C₅₋₂₀ carboarylgroup in which one aromatic carbon ring atom has been replaced by anaromatic nitrogen ring atom, then R³ can be any of the structures shownabove, where the benzene ring represents a C₅₋₂₀ carboaryl groupcontaining a nitrogen ring atom, for example:

[0128] where X is as defined above.

[0129] If the first group in R³ is a pyridyl group, rather than thephenyl group as illustrated above, the nitrogen ring atom may be at anyavailable position of the ring.

[0130] The first bridge may be situated at any possible position of thephenyl group in R³ and the optional second bridge group may be situatedon either adjacent atom of the phenyl group (if possible). Therefore,the resulting R³ group (as a whole) may be a radical at a number ofpossible positions on the benzene ring bound to the central moiety, forexample the following possible R³ group (unsubstituted xanthenyl):

[0131] may be a radical at the 1, 2, 3 or 4 positions.

Includes Other Forms

[0132] Included in the above are the well known ionic, salt, solvate,and protected forms of these substituents. For example, a reference tocarboxylic acid (—COOH) also includes the anionic (carboxylate) form(—COO⁻), a salt or solvate thereof, as well as conventional protectedforms. Similarly, a reference to an amino group includes the protonatedform (—N⁺HR¹R²), a salt or solvate of the amino group, for example, ahydrochloride salt, as well as conventional protected forms of an aminogroup. Similarly, a reference to a hydroxyl group also includes theanionic form (—O⁻), a salt or solvate thereof, as well as conventionalprotected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

[0133] Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; α- and β-forms; axial and equatorialforms; boat-, chair-, twist-, envelope-, and halfchair-forms; andcombinations thereof, hereinafter collectively referred to as “isomers”(or “isomeric forms”).

[0134] Note that, except as discussed below for tautomeric forms,specifically excluded from the term “isomers”, as used herein, arestructural (or constitutional) isomers (i.e. isomers which differ in theconnections between atoms rather than merely by the position of atoms inspace). For example, a reference to a methoxy group, —OCH₃, is not to beconstrued as a reference to its structural isomer, a hydroxymethylgroup, —CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to beconstrued as a reference to its structural isomer, metachlorophenyl.However, a reference to a class of structures may well includestructurally isomeric forms falling within that class (e.g., C₁₋₇ alkylincludes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, andtert-butyl; methoxyphenyl includes ortho-, meta-, andpara-methoxyphenyl).

[0135] The above exclusion does not pertain to tautomeric forms, forexample, keto-, enol-, and enolate-forms, as in, for example, thefollowing tautomeric pairs: keto/enol (illustrated below),imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

[0136] Note that specifically included in the term “isomer” arecompounds with one or more isotopic substitutions. For example, H may bein any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

[0137] Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g., fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

[0138] Unless otherwise specified, a reference to a particular compoundalso includes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below.

[0139] It may be convenient or desirable to prepare, purify, and/orhandle a corresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts”, J. Pharm. Sci., Vol. 66, pp. 1-19.

[0140] For example, if the compound is anionic, or has a functionalgroup which may be anionic (e.g., —COOH may be —COO⁻), then a salt maybe formed with a suitable cation. Examples of suitable inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺,alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such asAl³⁺. Examples of suitable organic cations include, but are not limitedto, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g.,NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substitutedammonium ions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

[0141] If the compound is cationic, or has a functional group which maybe cationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: acetic, propionic, succinic, glycolic, stearic, paimitic,lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic,pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,phenyisulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethanedisulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, andgluconic. Examples of suitable polymeric anions include, but are notlimited to, those derived from the following polymeric acids: tannicacid, carboxymethyl cellulose.

[0142] It may be convenient or desirable to prepare, purify, and/orhandle a corresponding solvate of the active compound. The term“solvate” is used herein in the conventional sense to refer to a complexof solute (e.g. active compound, salt of active compound) and solvent.If the solvent is water, the solvate may be conveniently referred to asa hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate,etc.

[0143] It may be convenient or desirable to prepare, purify, and/orhandle the active compound in a chemically protected form. The term“chemically protected form”, as used herein, pertains to a compound inwhich one or more reactive functional groups are protected fromundesirable chemical reactions, that is, are in the form of a protectedor protecting group (also known as a masked or masking group or ablocked or blocking group). By protecting a reactive functional group,reactions involving other unprotected reactive functional groups can beperformed, without affecting the protected group; the protecting groupmay be removed, usually in a subsequent step, without substantiallyaffecting the remainder of the molecule. See, for example, ProtectiveGroups in Organic Synthesis (T. Green and P. Wuts, Wiley, 1999).

[0144] For example, a hydroxy group may be protected as an ether (—OR)or an ester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl,benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; atrimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester(—OC(═O)CH₃, —OAc).

[0145] For example, an aldehyde or ketone group may be protected as anacetal or ketal, respectively, in which the carbonyl group (>C═O) isconverted to a diether (>C(OR)₂), by reaction with, for example, aprimary alcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid.

[0146] For example, an amine group may be protected, for example, as anamide or a urethane, for example, as: a methyl amide (—NHCO—CH₃); abenzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxy amide(—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulphonyl)ethyloxy amide (—NH-Psec); or, in suitable cases, asan N-oxide (>NO.).

[0147] For example, a carboxylic acid group may be protected as an esterfor example, as: an C₁₋₇ alkyl ester (e.g. a methyl ester; a t-butylester); a C₁₋₇ haloalkyl ester (e.g., a C₁₋₇ trihaloalkyl ester); atriC₁₋₇ alkylsilyl-C₁₋₇ alkyl ester; or a C₅₋₂₀ aryl-C₁₋₇ alkyl ester(e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example,as a methyl amide.

[0148] For example, a thiol group may be protected as a thioether (—SR),for example, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

[0149] It may be convenient or desirable to prepare, purify, and/orhandle the active compound in the form of a prodrug. The term “prodrug”,as used herein, pertains to a compound which, when metabolised (e.g. invivo), yields the desired active compound. Typically, the prodrug isinactive, or less active than the active compound, but may provideadvantageous handling, administration, or metabolic properties.

[0150] For example, some prodrugs are esters of the active compound(e.g. a physiologically acceptable metabolically labile ester). Duringmetabolism, the ester group (—C(═O)OR) is cleaved to yield the activedrug. Such esters may be formed by esterification, for example, of anyof the carboxylic acid groups (—C(═O)OH) in the parent compound, with,where appropriate, prior protection of any other reactive groups presentin the parent compound, followed by deprotection if required. Examplesof such metabolically labile esters include those wherein R is C₁₋₇alkyl (e.g. -Me, -Et); C₁₋₇ aminoalkyl (e.g. aminoethyl;2-(N,N-diethylamino)ethyl; 2-(4-morpholino) ethyl); and acyloxy-C₁₋₇alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl;acetoxymethyl; 1-acetoxyethyl;1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl; 1-(benzoyloxy)ethyl;isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl;cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl;cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl;(4-tetrahydropyranyloxy)carbonyloxymethyl;1-(4-tetrahydropyranyloxy)carbonyloxyethyl;(4-tetrahydropyranyl)carbonyloxymethyl; and1-(4-tetrahydropyranyl)carbonyloxyethyl).

[0151] Also, some prodrugs are activated enzymatically to yield theactive compound, or a compound which, upon further chemical reaction,yields the active compound. For example, the prodrug may be a sugarderivative or other glycoside conjugate, or may be an amino acid esterderivative.

Further Preferences

[0152] The following preferences may be different for different aspectsof the present invention, and may be combined together.

[0153] In compounds of formula I, it is preferred that P is O and Q isCH, i.e. that the compound is of formula Ia.

[0154] Y is preferably O.

[0155] In formula I, when R¹ and R² form, along with the nitrogen atomto which they are attached, a heterocyclic ring having from 4 to 8atoms, this may form part of a C₄₋₂₀ heterocyclyl group defined above(except with a minimum of 4 ring atoms), which must contain at least onenitrogen ring atom. It is preferred that R¹ and R² form, along with thenitrogen atom to which they are attached, a heterocyclic ring having 5,6 or 7 atoms, more preferably 6 ring atoms.

[0156] Single rings having one nitrogen atom include azetidine,azetidine, pyrrolidine (tetrahydropyrrole), pyrroline (e.g.,3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (isopyrrole,isoazole), piperidine, dihydropyridine, tetrahydropyridine, and azepine;two nitrogen atoms include imidazolidine, pyrazolidine (diazolidine),imidazoline, pyrazoline (dihydropyrazole), and piperazine; one nitrogenand one oxygen include tetrahydrooxazole, dihydrooxazole,tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine,dihydrooxazine, and oxazine; one nitrogen and one sulphur includethiazoline, thiazolidine, and thiomorpholine.

[0157] Preferred rings are those containing one heteroatom in additionto the nitrogen, and in particular, the preferred heteroatoms are oxygenand sulphur. Thus preferred groups include morpholino, thiomorpholino,thiazolinyl. Preferred groups without a further heteroatom includepyrrolidino.

[0158] The most preferred groups are morpholino and thiomorpholino.

[0159] As mentioned above, these heterocyclic groups may themselves besubstituted; a preferred class of substituent is a C₁₋₇ alkyl group.When the heterocyclic group is morpholino, the substituent group orgroups are preferably methyl or ethyl, and more preferably methyl. Asole methyl substituent is most preferably in the 2 position.

[0160] As well as the single ring groups listed above, rings withbridges or cross-links are also envisaged. Examples of these types ofring where the group contains a nitrogen and an oxygen atom are:

[0161] These are named 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl,6-oxa-3-aza-bicyclo[3.1.0]hex-3-yl, 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl,and 7-oxa-3-aza-bicyclo[4.1.0]hept-3-yl, respectively.

[0162] In R³, the phenyl or pyridyl group is preferably a phenyl group.

[0163] R^(C1) and R^(C2) are preferably H.

[0164] R^(N) is preferably H, or an ester.

[0165] Preferred substituents of the phenyl or pyridyl ring in R³include, but are not limited to, halo, hydroxy, C₁₋₇ alkyl, C₁₋₇ alkoxy,acyl, acyloxy, amino, nitro, cyano, thiol and C₁₋₇ alkylthio, with haloand hydroxy being most preferred.

[0166] Preferred substituents of the phenyl or pyridyl ring or the C₅₋₂₀carboaryl group in R³ also include, but are not limited to, acylamido,sulfonamino, ether, ester, amido, amino and acyl.

[0167] In the acylamido group, the amide substituent is preferablyhydrogen, and the acyl substituent is preferably selected from ester(where the ester substituent is alkyl or aryl), C₁₋₇ alkyl (optionallysubstituted by ether, ester, C₅₋₂₀ aryl, acyloxy, amino andheterocyclyl), C₅₋₂₀ aryl (optionally substituted by alkoxy, alkyl,alkoxy, ester and C₅₋₂₀ aryl) and C₃₋₂₀ heterocylyl (optionallysubstitued by acyl)

[0168] A particularly preferred acyl susbtituent on the acylamido groupis of formula III:

[0169] wherein n is 1 to 4, preferably 1 or 2, and R³ and R⁴ areindependently hydrogen, an optionally substituted C₁₋₇ alkyl group,C₃₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or may together form,along with the nitrogen atom to which they are attached, an optionallysubstituted heterocyclic ring having from 4 to 8 ring atoms.

[0170] In the sulfonamino group, the amino substituent is preferablyhydrogen and the sulfonamino substituent selected from C₁₋₇ alkyl andC₅₋₂₀ aryl.

[0171] In the ether group, the ether substituent is preferably C₁₋₇alkyl (optionally substituted by amino, C₃₋₂₀ heterocyclyl, thioetherand C₅₋₂₀ aryl). A particularly preferred ether substituent is offormula III (defined above).

[0172] In the amido group, the amido substituents are preferablyindependently selected from hydrogen and C₁₋₇ alkyl (optionallysubstituted by C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl and amino). A particularlypreferred amido substituent is of formula III (defined above).

[0173] In the acyl group, the acyl substituent is preferably C₃₋₂₀heterocyclyl.

[0174] These substituents are preferably either para to the radicalposition in the phenyl or pyridyl group, and when the first bridge groupis ortho the radical position in the phenyl or pyridyl group, para tothe first bridge group in the C₅₋₂₀ aryl group (especially when thatgroup is phenyl).

[0175] Preferred structures for R³ include, but are not limited to thefollowing ‘core’ groups, which may bear substitution at appropriatepositions, where * indicates the preferred radical position (which istypically adjacent the first bridge group on the phenyl group):

[0176] with the most preferred core structures being:

[0177] Particularly preferred R³ groups include:

[0178] wherein R stands for the appropriate substituent group, asdefined above.

Acronyms

[0179] For convenience, many chemical moieties are represented usingwell known abbreviations, including but not limited to, methyl (Me),ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), tert-butyl(tBu), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh),benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz),acetyl (Ac), 1,3-bis(diphenylphosphino)propane (dppf).

[0180] For convenience, many chemical compounds are represented usingwell known abbreviations, including but not limited to, methanol (MeOH),ethanol (EtOH), iso-propanol (i-PrOH), methyl ethyl ketone (MEK), etheror diethyl ether (Et₂O), acetic acid (AcOH), dichloromethane (methylenechloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF),tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).

Synthesis Routes

[0181] Compounds according to the first aspect of the invention, offormula Ia, where Y═O, may be synthesised by the coupling of a2-chloro-6-amino-pyran-4-one to an appropriate arylboronic acid orarylboronate ester using a palladium catalysed coupling reaction, e.g.Suzuki coupling. Compounds where Y═S can be derived from thecorresponding compound where Y═O.

Synthesis of 2-chioro-6-amino-pyran-4-ones

[0182] These may be synthesised by the following route:

[0183] In step (a) CCl₄ is added across the carbon-carbon double bond ofdiketene by free-radical addition to yield4-chloro-4(2,2,2,-trichloro-ethyl)-oxetan-2-one (1). Suitable initiatorsinclude peroxide, such as BCHPO((bis-4-t-butylcyclohexyl)peroxydicarbonate).

[0184] In step (b), the amine R¹R²NH opens the lactone ring bynucleophilic attack at the carbonyl centre. The oxy anion generated thendisplaces the chlorine atom on the α-carbon to give rise to aβ-keto-amide intermediate. Further elimination of HCl finally give the5,5-dichloro-1-amino-pent-4-ene-1,3-dione. Suitable conditions for thisstep include inorganic base such as sodium hydrogen carbonate andsolvent such as dry dichloromethane.

[0185] In step (c), ring closure takes place by displacement of one ofthe 5-chloro groups by the oxygen of the amide moiety to form thepyran-4-one ring, which reaction is catalysed by a Lewis acid, such asperchloric acid.

Arylboronic Acids and Arylboronate Esters

[0186] Some appropriate arylboronic acids and arylboronate esters arecommercially available. Other appropriate arylboronic acids andarylboronate esters may be synthesised by using one of the followingroutes, in which the starting materials are commercially available orreadily synthesised. For example, a synthesis route to thioxanthenone isdescribed in Archer, S., et al., J. Med. Chem., 25, 220-227, 1982, andthe conversion of thioxanthenone to thiothanxene is described inMlotkowska, B. L., et al., J. Heterocyclic Chem., 28, 731-736, 1991.Other routes are shown in the examples, and include routes where thecentral C₅₋₇ ring is synthesised by ring closure from an appropriatecarboxylic acid, optionally followed by reduction of the remaining ketogroup.

[0187] Synthesis of Aryl Boronate Esters

[0188] (a): PdCl2dppf, dppf, Pinacol diborane, KOAc

[0189] where R is the remainder of the R³ group

[0190] Aryl boronate esters may be formed by Pd(0)-catalysed crosscoupling reaction of the appropriate aryl triflate or aryl halide withtetra(alkoxy)diboron, e.g. pinacol diboron. Suitable conditions includethe use of a catalyst, such as PdCl₂dppf, extra ligands, such as dppf,potassium acetate as a base, in a solvent such as dioxane, DMF or DMSO.

[0191] Examples of this method are to be found in T Ishiyama, et al.,Tet. Lett., vol. 38, no. 19, 3447-3450, 1997 and A Giroux, et al., Tet.Lett., vol. 38, no. 22, 3841-3844, 1997.

[0192] Synthesis of Aryl Boronic Acids

[0193] (a):t-BuLi, (EtO)₃B

[0194] where R is the remainder of the R³ group

[0195] Boronic acids may be generated via lithiation of the aromaticring by tert-butyl lithium followed by the reaction of the anion formedwith alkyl borate such as triethyl borate to give the desired arylboronic acid.

Palladium Catalysed Coupling

[0196] The coupling of the arylboronic acid or arylboronate ester to the2-chloro-6-amino-pyran-4-one can be carried out using the normalconditions, e.g. a palladium catalyst (Pd(PPh₃)₄, Pd(dppf)Cl₂) and base(Na₂CO₃, NaOCH₂CH₃, TlOH, N(CH₂CH₃)₃, K₃PO₄).

[0197] Compounds according to the first aspect of the invention, offormula Ib, where Y═O may be synthesised according to the followingmethod, wherein R represents the rest of R³:

[0198] In step (a), CS₂ is added to the acetophenone derivative, in thepresence of a base, such as potassium tert-butoxide, to yield a3-aryl-3-hydroxy-dithioacrylic acid.

[0199] In step (b), iodoethane undergoes nucleophiic attack by theactivated thioacid, to yield the ethyl ester. Activation of the thioacidcan be achieved by the use of base, for example, a mixture oftetrabutylammonium hydrogen sulphate and sodium hydroxide.

[0200] In step (c), an amine displaces the ethyl group, which isfollowed in step (d) by reaction of the remaining thio group withiodoethane (via the tautomeric compound).

[0201] The final step (e) is a condensation with ethyl bromoacetate toyield the ring-closed 4-amino-6-aryl-pyran-2-one.

Conversion of Y from O to S

[0202] This conversion may be achieved using Lawesson's reagent in anorganic solvent, such as toluene, followed by the appropriatepurification steps. Protection of groups sensitive to Lawesson's reagentcan be carried out before it is used, followed by deprotection once thepyranthione has been synthesised.

Use of Compounds of the Invention

[0203] The present invention provides active compounds, specifically,active 2-aryl-6-amino-pyran-4-ones, 2-aryl-6-amino-pyran-4-thiones,4-amino-6-aryl-pyran-2-ones and 4-amino-6-aryl-pyran-2-thiones.

[0204] The term “active”, as used herein, pertains to compounds whichare capable of inhibiting ATM activity, and specifically includes bothcompounds with intrinsic activity (drugs) as well as prodrugs of suchcompounds, which prodrugs may themselves exhibit little or no intrinsicactivity.

[0205] One assay which may be used in order to assess the ATM inhibitionoffered by a particular compound is described in the examples below.

[0206] The present invention further provides a method of inhibiting ATMin a cell, comprising contacting said cell with an effective amount ofan active compound, preferably in the form of a pharmaceuticallyacceptable composition. Such a method may be practised in vitro or invivo.

[0207] For example, a sample of cells (e.g. from a tumour) may be grownin vitro and an active compound brought into contact with said cells inconjunction with agents that have a known curative effect, and theenhancement of the curative effect of the compound on those cellsobserved.

[0208] The present invention further provides active compounds whichinhibit ATM activity as well as methods of inhibiting ATM activitycomprising contacting a cell with an effective amount of an activecompound, whether in vitro or in vivo.

[0209] The invention further provides active compounds for use in amethod of treatment of the human or animal body. Such a method maycomprise administering to such a subject a therapeutically-effectiveamount of an active compound, preferably in the form of a pharmaceuticalcomposition.

[0210] The term “treatment” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g. in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure (i.e.prophylaxis) is also included.

[0211] The term “therapeutically-effective amount” as used herein,pertains to that amount of an active compound, or a material,composition or dosage form comprising an active compound, which iseffective for producing some desired therapeutic effect, commensuratewith a reasonable benefit/risk ratio.

[0212] The present inventors have found that compounds of the presentinvention can efficiently repress retroviral vector transduction inone-step, cell based integration assays (termed LUCIA) and inhibit HIV-1infection in 4-day replication assays at sub-micromolar concentrations.Further, in contrast to the observations of Daniel et al., where it wasconcluded that the effect of ATM on retroviral integration would only beseen in a DNA-PK-deficient background, this effect works in the presenceof functional DNA-PK activity.

[0213] Initial linkage of linear retroviral DNA with host cellchromosomal DNA is catalysed by viral integrase (IN) and results inshort staggered DNA strand breaks in the host cell DNA at the site ofattachment (Brown, P. O. (1990) Integration of retroviral DNA. Curr TopMicrobiol Immunol, 157, 19-48). These gapped DNA intermediates are shownto be sensed as sites of DNA damage by the host cell and repaired by theATM pathway to complete the process of integration and allow productiveinfection to occur. Compounds of the invention prevent the repair ofgapped DNA intermediates by the ATM pathway and thus prevent completeintegration of retroviral DNA into the host genome.

[0214] As described above, the invention provides a compound as definedin the first aspect of the invention for use in the treatment ofretroviral infection and the use of such a compound in the manufactureof a medicament for use in the treatment of retroviral infection.

[0215] Also provided by the invention is a method of treatment of aretroviral infection comprising administering a compound as defined inthe first aspect of the invention to an individual in need thereof.

[0216] An exemplary compound of the invention which is shown to beuseful in the treatment of retroviral infection is2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4-one (4).

[0217] Retroviral mediated diseases which may be treated as describedabove include HIV infection and acquired immunodeficiency syndrome(AIDS) and Human T-cell Leukaemia virus (HTLV) infection and itsassociated diseases adult T-cell leukaemia/lymphoma (ATLL) and tropicalspastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM).

[0218] Compounds of the invention may be used in combination with otherretroviral therapies to suppress virus replication, for example in a‘highly active anti-retroviral therapy’ or HAART treatment.

[0219] The invention provides a pharmaceutical composition comprising acompound as described herein and one or more other anti-retroviralagents.

[0220] The invention also provides a composition comprising a compoundas defined in the first aspect of the invention and one or more otheranti-retroviral agents for treatment of a retroviral infection and theuse of such a composition in the manufacture of a medicament for use inthe treatment of a retroviral infection.

[0221] Suitable anti-retroviral agents which inhibit retroviralreplication, for example retroviral protease inhibitors (PI) such asSequinavir, Indinavir, Ritonavir and Nelfinavir, nucleoside retroviralreverse transcriptase inhibitors such as 3′-azido-3′deoxythymidine (AZT;Zidovudine), 2′,3′-Dideoxycytosine (ddC; Zalcitabine),2′,3′-Dideoxyinosine (ddI; Didanosine)and 3TC; (Lamivudine), andnon-nucleoside retroviral reverse transcriptase inhibitors such asNevirapine, Delavirdine and Efavirenz.

Administration

[0222] The active compound or pharmaceutical composition comprising theactive compound may be administered to a subject by any convenient routeof administration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly.

[0223] The subject may be a eukaryote, an animal, a vertebrate animal, amammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

Formulations

[0224] While it is possible for the active compound to be administeredalone, it is preferable to present it as a pharmaceutical composition(e.g. formulation) comprising at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

[0225] Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilisers, or other materials, asdescribed herein.

[0226] The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

[0227] Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0228] The formulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

[0229] Formulations may be in the form of liquids, solutions,suspensions, emulsions, elixirs, syrups, tablets, losenges, granules,powders, capsules, cachets, pills, ampoules, suppositories, pessaries,ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils,boluses, electuaries, or aerosols.

[0230] Formulations suitable for oral administration (e.g. by ingestion)may be presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active compound; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion; as a bolus; as an electuary; or as apaste.

[0231] A tablet may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g. povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g. lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc, silica);disintegrants (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g. sodium lauryl sulfate); andpreservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid). Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activecompound therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

[0232] Formulations suitable for topical administration (e.g.transdermal, intranasal, ocular, buccal, and sublingual) may beformulated as an ointment, cream, suspension, lotion, powder, solution,past, gel, spray, aerosol, or oil. Alternatively, a formulation maycomprise a patch or a dressing such as a bandage or adhesive plasterimpregnated with active compounds and optionally one or more excipientsor diluents.

[0233] Formulations suitable for topical administration in the mouthinclude losenges comprising the active compound in a flavoured basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive compound in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active compound in asuitable liquid carrier.

[0234] Formulations suitable for topical administration to the eye alsoinclude eye drops wherein the active compound is dissolved or suspendedin a suitable carrier, especially an aqueous solvent for the activecompound.

[0235] Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of about 20 to about 500 microns which isadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable formulations wherein the carrier is aliquid for administration as, for example, nasal spray, nasal drops, orby aerosol administration by nebuliser, include aqueous or oilysolutions of the active compound.

[0236] Formulations suitable for administration by inhalation includethose presented as an aerosol spray from a pressurised pack, with theuse of a suitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichorotetrafluoroethane, carbon dioxide, orother suitable gases.

[0237] Formulations suitable for topical administration via the skininclude ointments, creams, and emulsions. When formulated in anointment, the active compound may optionally be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activecompounds may be formulated in a cream with an oil-in-water cream base.If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the active compoundthrough the skin or other affected areas. Examples of such dermalpenetration enhancers include dimethylsulfoxide and related analogues.

[0238] When formulated as a topical emulsion, the oily phase mayoptionally comprise merely an emulsifier (otherwise known as anemulgent), or it may comprises a mixture of at least one emulsifier witha fat or an oil or with both a fat and an oil. Preferably, a hydrophilicemulsifier is included together with a lipophilic emulsifier which actsas a stabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

[0239] Suitable emulgents and emulsion stabilisers include Tween 60,Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearateand sodium lauryl sulphate. The choice of suitable oils or fats for theformulation is based on achieving the desired cosmetic properties, sincethe solubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono-or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required.

[0240] Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

[0241] Formulations suitable for rectal administration may be presentedas a suppository with a suitable base comprising, for example, cocoabutter or a salicylate.

[0242] Formulations suitable for vaginal administration may be presentedas pessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

[0243] Formulations suitable for parenteral administration (e.g. byinjection, including cutaneous, subcutaneous, intramuscular, intravenousand intradermal), include aqueous and non-aqueous isotonic,pyrogen-free, sterile injection solutions which may containanti-oxidants, buffers, preservatives, stabilisers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents, and liposomesor other microparticulate systems which are designed to target thecompound to blood components or one or more organs. Examples of suitableisotonic vehicles for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active compound in the solution is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.Formulations may be in the form of liposomes or other microparticulatesystems which are designed to target the active compound to bloodcomponents or one or more organs.

Dosage

[0244] It will be appreciated that appropriate dosages of the activecompounds, and compositions comprising the active compounds, can varyfrom patient to patient. Determining the optimal dosage will generallyinvolve the balancing of the level of therapeutic benefit against anyrisk or deleterious side effects of the treatments of the presentinvention. The selected dosage level will depend on a variety of factorsincluding, but not limited to, the activity of the particular compound,the route of administration, the time of administration, the rate ofexcretion of the compound, the duration of the treatment, other drugs,compounds, and/or materials used in combination, and the age, sex,weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, although generally thedosage will be to achieve local concentrations at the site of actionwhich achieve the desired effect without causing substantial harmful ordeleterious side-effects.

[0245] Administration in vivo can be effected in one dose, continuouslyor intermittently (e.g. in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician.

[0246] In general, a suitable dose of the active compound is in therange of about 100 μg to about 250 mg per kilogram body weight of thesubject per day. Where the active compound is a salt, an ester, prodrug,or the like, the amount administered is calculated on the basis of theparent compound and so the actual weight to be used is increasedproportionately.

EXAMPLES

[0247] The following examples are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein.

[0248] In these examples, reference is made to the following figures.

[0249]FIG. 1 shows that compound 4 can sensitise cells to ionisingradiation. The % survival of HeLa cells was measured with increasingionising radiation, in the absence of compound 4 (▪), and at twodifferent concentrations of compound 4, 0.5 μM (▾) and 2 μM ().

[0250]FIG. 2 shows that compound 4 can sensitise cells to etopside. The% survival of LoVo cells was measured with increasing concentrations ofetopside, in the absence of compound 4 (▪), and in the presence of 10 μMof compound 4 ()

[0251]FIG. 3 shows that compound 4 can sensitise cells to camptothecin.The % survival of LoVo cells was measured with increasing concentrationsof camptothecin, in the absence of compound 4 (▪), and in the presenceof 10μM of compound 4 ().

[0252]FIG. 4 shows that compound 4 can sensitise cells to doxorubicin.The % survival of LoVo cells was measured with increasing concentrationsof doxorubicin, in the absence of compound 4 (▪), and in the presence of10 μM of compound 4 ().

[0253]FIG. 5 shows that compound 4 can inhibit recombinant retroviralvector infections. The inhibition of retroviral transduction by the ATMinhibitor Compound 4 was assessed by performing HIV-1 based LUCIA onJurkat T-cells in the presence of increasing concentrations of compound4 (♦). Data are presented as transduction efficiency (luciferase signal)relative to untreated control cells. The IC₅₀ concentration for HIV-1infections by compound 4 is around 1 μM. Drug cytotoxicity (□) wasdetermined by MTS formazan dye reduction assays and data are presentedas the percentage of viable cells remaining after drug treatment. Nosignificant cytotoxicity was observed over the concentration range ofCompound 4 tested.

[0254]FIG. 6 shows that Compound 4 does not inhibit HIV-1 RT. Theinhibition of HIV-1 RT was assessed by performing chemilluminescentHIV-1 reverse transcriptase assays in the presence of increasing amountsof compound 4 (♦). No significant anti-RT activity for compound 4 isobserved over the concentration range used. Control RT inhibition usingnevirapine (□) is also shown.

[0255]FIG. 7 shows that Compound 4 acts synergistically with AZT toinhibit HIV-1 infections. HIV-1 based LUCIA was performed on HeLa cellswith increasing concentrations of Compound 4 in the absence (▾) orpresence of 0.1 μM (□), 0.4 μM (*) or 1.2 μM (⋄) AZT. Data are presentedas transduction efficiency (as determined by luciferase activity)relative to untreated control cells. The combined presence of bothCompound 4 and AZT shows enhanced anti-HIV activity when compared toeach drug alone.

[0256]FIG. 8 shows that Compound 4 inhibits HIV-1 replication. 4-dayHIV-1 replication assays were performed on C1866 cells in the presenceof increasing concentrations of Compound 4 (♦) or AZT (□). HIV-1 titreswere quantified by p24 antigen ELISA and data are shown as thepercentage of HIV-1 p24 in cell-free supernatants relative to untreatedcontrol cells. (A) Replication assays performed using wild type HIV-1strain (HIV-1_(HXB2) wt). (B) Replication assays performed using an AZTresistant HIV-1 strain (HIV-1_(HXB2) AZTres). Compound 4 inhibits HIV-1replication equally well in both wild-type and AZT resistant HIV-1strains. (C) Control drug cytotoxicity (Δ) was determined by XTT dyereduction assays. Data are presented as the percentage of viable cellsremaining after drug treatment. No significant cytotoxicity was observedover the effective Compound 4 concentration range shown to inhibit HIV-1replication.

A) Chemical Examples General Experimental Methods

[0257] Thin layer chromatography was carried out using Merck Kieselgel60 F₂₅₄ glass backed plates. The plates were visualized by the use of aUV lamp (254 nm). Silica gel 60 (particle sizes 40-63μ) supplied by E.M. Merck was employed for flash chromatography. ¹H NMR spectra wererecorded at 300 MHz on a Bruker DPX-300 instrument. Chemical shifts werereferenced to tetramethylsilane.

[0258] Purification and Identification of Libraries Samples

[0259] The samples were purified on Gilson LC units.

[0260] Mobile phase A-0.1% aqueous TFA, Mobile phase B-Acetonitrile,Flow rate 6 ml/min., Gradient-typically starting at 90% A/10% B for oneminute, rising to 97% B after 15 minutes, holding there for 2 minutes,then back to the starting conditions. Column: Jones ChromatographyGenesis 4μ C18 column, 10 mm×250 mm. Peak acquisition based on UVdetection at 254 nm.

[0261] Mass Specs were recorded on a Finnegan LCQ instrument in positiveion mode.

[0262] Mobile phase A-0.1% aqueous formic acid, Mobile phaseB-Acetonitrile, Flow rate 2 ml/min., Gradient -starting at 95% A/5% Bfor one minute, rising to 98% B after 5 minutes, holding there for 3minutes, then back to the starting conditions. Column-Phenomenex 5μ LunaC18 column, 4.6 mm×50 mm UV detection at 254 nm, PDA detection scanningfrom 210 to 600 nm.

[0263] Mass Spectra of Other Compounds

[0264] Mass spectra of-non-library compounds and building blocks wererecorded on a Micromass ZQ instrument (single quadrupole, operating inelectrospray ionisation mode), using a Waters 600 HPLC Pump and 2700Autosampler.

[0265] Mobile Phase A: 0.1% Formic acid in water, Mobile phase B: 0.1%Formic acid in acetonitrile, Flow rate: 2.0 ml/min., Gradient: 5% B to95% B over 3 mins, hold 3 mins. Column: Varies, but always C18 50 mm×4.6mm (Currently Genesis C18 4μ. Jones Chromatography). PDA detection:Waters 996, scan range 210-400 nm.

Synthesis of 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)

[0266]

4-Chloro-4-(2,2,2-trichloro-ethyl)-oxetan-2-one (1)

[0267] A solution of BCHPO (bis-4-t-butylcyclohexyl)peroxydicarbonate(11.8 g) and diketene (83.5 ml) in CCl₄ (300 ml) was added dropwise over120 minutes to a refluxing solution of CCl₄, and was stirred for afurther 1 hour. The resulting pale yellow solution was cooled andazeotroped with DCM. The resulting residue was stirred with hexane(3×150 ml) for 10 minutes and the liquor was decanted off through acelite pad. The filtered liquors were combined and concentrated in vacuoto give 1 as a pale yellow oil (125.0 g, 52.9%).

5,5-Dichloro-1-morpholin-4-yl-pent-4-ene-1,3-dione (2)

[0268] Two separate solutions of 1 (62.5 g, 0.26 mmol) and morpholine(24.0 g, 0.28 mol) in DCM (120 ml) were added simultaneously to amixture of NaHCO₃ (44.0 g, 0.52 mol) in dry DCM (300 ml). The reactionwas maintained at 15° C. over 140 minutes with stirring. The reactionwas filtered, washed with DCM (3×100 ml) and the combined organic layerswere concentrated in vacuo to a slurry which was then passed through ashort silica pad, and further washed with DCM (4×100 ml). The combinedorganic layers were concentrated in vacuo, suspended in hexane (400 ml)and stirred for 1 hour, filtered and dried to give a cream solid. Thesolid was suspended in TBME (100 ml), stirred for 15 minutes, filtered,washed with TBME and dried to give 2 as a white powder (47.8 g, 72%).m/z (LC-MS, ESP): 252 (M⁺+1).

2-Chloro-6-morpholin-4-yl-pyran-4-one (3)

[0269] To a suspension of 2 (11.3 g, 44.9 mmol) in dioxane was addedperchloric acid (11.4 ml, 0.14 mol) and the reaction was heated at 90°C. under N₂ for 1 hour. The reaction was cooled, neutralised with 2MNaOH (75 ml) and filtered. The aqueous layer was extracted with DCM(4×30 ml) and the organic layers were combined and dried over MgSO₄. Theorganic layer was further treated with charcoal and filtered throughcelite. The dark yellow filtrate was evaporated in vacuo, and theresulting solid was triturated with hexane (50 ml) and dried to give 3(7.3 g, 75%) as a light yellow powder. m/z (LC-MS, ESP): 216 (M⁺+1).¹H-NMR (300 MHz, DMSO-d₆): 3.3 (t, 4H), 3.65 (t, 4H), 5.4 (d, 1H), 6.25(d, 1H).

Example 1 Synthesis of 2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4-one(4)

[0270]

[0271] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (863 mg, 4 mmol),thianthrene-1-boronic acid (1.145 g, 4.4 mmol), and ground potassiumcarbonate (1.105 g, 8 mmol) were suspended in dioxane (10 ml) anddegassed (sonication for 5 minutes then saturated with N₂). Pd(PPh₃)₄(231 mg, 0.2 mmol) was then added and the reaction mixture was thenheated at 90° C. for 24 hours under a vigorous stirring and a N₂atmosphere. The solvent was removed in vacuo and the residue was thensuspended in water 50 ml) and extracted with ethyl acetate (3×100 ml).The organics were combined, washed with saturated brine and dried oversodium sulphate. The solvent was removed in vacuo and the residue waspurified by column chromatography (silica; ethyl acetate:ethanol; 9:1)to give the title compound as a white solid (70 mg, 4%). ¹H-NMR (300MHz, DMSO-d₆): δ_(H)=3.44 (4H, t, J 5 Hz); 3.76 (4H, t, J 5 Hz); 5.57(1H, d, J 2 Hz); 6.30 (1H, d, J 2 Hz); 7.43 (2H, m); 7.53 (1H, t, 8 Hz);7.66 (3H, m); 8.49 (1H, dd, J land 8 Hz). m/z (LC-MS, ESP): 396 (M⁺+1).

Example 2 Synthesis of 2-Phenoxathiin-4-yl-6-morpholin-4-yl-pyran-4-one(5)

[0272]

[0273] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(863 mg, 4 mmol),phenoxathiin-4-boronic acid (1.07 g, 4.4 mmol), and ground potassiumcarbonate (1.1 g, 8 mmol) were suspended in dioxane (10 ml) and degassed(sonication for 5 minutes then saturated with N₂). Pd(PPh₃)₄ (231 mg,0.2 mmol) was then added and the reaction mixture was then heated at 90°C. for 24 hours under a vigorous stirring and a N₂ atmosphere. Thesolvent was removed in vacuo and the residue was then suspended in water(50 ml) and extracted with ethyl acetate (3×50 ml). The organics werecombined, washed with saturated brine and dried over sodium sulphate.The solvent was removed in vacuo and the residue was purified by columnchromatography (silica; ethyl acetate:ethanol; 9:1) to give the titlecompound as a white solid (620 mg, 41%). ¹H-NMR (300 MHz, DMSO-d₆):δ=3.38 (4H, t, J 5 Hz); 3.71 (4H, t, J 5 Hz); 5.49 (1H, d, J 2 Hz); 6.49(1H, d, J 2 Hz); 7.06 (1H, dd, J 1 and 8 Hz); 7.26 (4H, m); 7.46 (1H,dd, J 1.5 and 8 Hz); 7.55 (1H, dd, J 1.5 and 8 Hz). m/z (LC-MS, ESP):380 (M⁺+1).

Example 3 Synthesis of 2-Dibenzofuran-1-yl-6-morpholin-4-yl-pyran-4-one(6)

[0274]

[0275] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(22 mg, 0.1 mmol),4-dibenzofuran-1-boronic acid (28 mg, 0.13 mmol), and caesium carbonate(65 mg, 0.2 mmol) were suspended in dioxane (0.5 ml) and degassed(sonication for 5 minutes then saturated with N₂). Pd(PPh₃)₄ (5 mg,0.005 mmol) was then added and the reaction mixture was then heated at90° C. for 24 hours under a vigorous stirring and a N₂ atmosphere. Thereaction mixture was purified by preparative HPLC to give the titlecompound (2.1 mg; 6%). m/z (LC-MS, ESP): 348 (M⁺+1).

Example 4 Synthesis of2-Dibenzothiophen-1-yl-6-morpholin-4-yl-pyran-4-one (7)

[0276]

[0277] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(740 mg, 3.43 mmol),dibenzothiophene-1-boronic acid (860 mg, 3.77 mmol), and groundpotassium carbonate (964 mg, 6.86 mmol) were suspended in dioxane (10ml) and degassed (sonication for 5 minutes then saturated with N₂).Pd(PPh₃)₄ (200 mg, 0.17 mmol) was then added and the reaction mixturewas then heated at 90° C. for 24 hours under a vigorous stirring and aN₂ atmosphere. The solvent were removed in vacuo and the residue wasthen suspended in water (50 ml) and extracted with ethyl acetate (3×50ml). The organics were combined, washed with saturated brine and driedover sodium sulphate. The solvent was removed in vacuo and the residuewas purified by column chromatography (silica; ethyl acetate:ethanol;9:1) to give the title compound as a white solid (80 mg, 6%). ¹H-NMR(300 MHz, DMSO-d₆): δ_(H)=3.49 (4H, t, J 5 Hz); 3.76 (4H, t, J 5 Hz);5.53 (1H, d, J 2 Hz); 6.63 (1H, d, J 2 Hz); 7.59 (2H, m); 7.69 (1H, t, J8 Hz); 7.96 (1H, dd, J 1 and 7.5 Hz); 8.11 (1H, m); 8.47 (1H, m); 8.57(1H, dd, J 1 and 8 Hz). m/z (LC-MS, ESP): 364 (M⁺+1).

Example 5 Synthesis of2-(2-Phenylsulfanyl-phenyl)-6-morpholin-4-yl-pyran-4-one (9)

[0278] (a) 2-Phenylsulfido-benzene Boronic Acid (8)

[0279] To a cooled (−78° C.), stirred solution of diphenyl sulphide(1.66 ml, 10 mmol) in 30ml anhydrous THF, was added dropwise under anitrogen atmosphere 7 ml t-BuLi. Upon addition of t-BuLi the solutionturned orange then brown. The mixture was allowed to warm to roomtemperature and then left stirring for 3 hours. The mixture was cooled(−78° C.). Triethyl borate (2.03 ml, 12 mmol) was added dropwise to thecooled yellow solution turning the solution lime coloured. During thisaddition, the temperature was monitored and not allowed to rise above−75° C. The solution was then left to warm to room temperature and leftstirring for 2 hours. Water was added to the reaction mixture and theaqueous were extracted with diethyl ether. The aqueous layer (pH 14) wasacidified to pH 1 with (1 M HCl) and the product was extracted intodiethyl ether. The organics were dried over magnesium sulphate and theorganics were evaporated off in vacuo, yielding an oily residue (690 mg,30%), which was used without further purification.

[0280] (b) 2-(2′-Phenylsulfido-phenyl)-6-morpholin-4-yl-pyran-4-one (9)

[0281] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(582 mg, 2.7 mmol),2-phenylsulphido-benzene boronic acid (8) (690 g, 3 mmol), and groundpotassium carbonate (819 mg, 5.94 mmol) were suspended in dioxane (10ml) and degassed (sonication for 5 minutes then saturated with N₂).Pd(PPh₃)₄ (156 mg, 0.13 mmol) was then added and the reaction mixturewas then heated at 90° C. for 24 hours under a vigorous stirring and aN₂ atmosphere. The solvent was removed in vacuo and the residue waspurified by preparative HPLC to give the title compound (27 mg, 3%).¹H-NMR (300 MHz, DMSO-d₆): δ_(H)=3.37 (4H, t); 3.76 (4H, t) 5.45 (1H,d); 6.31 (1H, d); 7.32-7.55 (9H, m). m/z (LC-MS, ESP): 366 (M⁺+1).

Example 6 Synthesis of2-(1-Fluoro-9-oxo-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (13)

[0282]

[0283] a: H₂SO₄, Thiosalicylic acid; b: Tf₂O, pyridine; c:bis(pinacolato)diboron, PdCl₂dppf, dppf, dioxane,100° C.; d:chloropyranone, Pd(PPh₃)₄, dioxane, 90° C.

[0284] (a) 1-Fluoro-4-hydroxy-thioxanthen-9-one (10)

[0285] Thiosalicylic acid (46.26 g, 0.3 mol) and 4-fluorophenol (56.05g, 0.5 mol) were dissolved in conc. H₂SO₄ (750 ml) and the mixture wasstirred under nitrogen for 24 hours. The reaction mixture was thenpoured onto ice (1.5 L) and the yellow precipitate was filtered andwashed with water (300 ml). The precipitate was dried at 50° C. for 24hours and was used without further purification (31.4 g, 42.5%). m/z(LC-MS, ESP): 247 (M⁺+1).

[0286] (b) 1-Fluoro-9-oxo-thioxanthen-4-yl Trifluoromethane Sulfonate(11)

[0287] 1-Fluoro-4-hydroxy-thioxanthen-9-one (4.92 g, 20 mmol) wasdissolved in dry pyridine (100 ml) and cooled to 0° C. under a nitrogenatmosphere. Triflic anhydride (3.66 ml, 22.3 mmol) was added drop wiseto the stirred solution over 5 minutes. The reaction was left overnightand was then poured onto water (300 ml) and the precipitate formed wasfiltered. The solid was purified through a plug of silica (ethylacetate:hexane; 1:9) to give the title compound as a white fluffy solid(1.72 g, 22.4%). m/z (LC-MS, ESP): 379 (M⁺+1).

[0288] (c)1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-thioxanthen-9-one(12)

[0289] 1-fluoro-9-oxo-thioxanthen-4-yl trifluoromethane sulfonate (11)(378 mg, 1 mmol), bis(pinacolato)diboron (305 mg, 1.2 mmol), and groundpotassium acetate (294 mg, 3mmol) were suspended in dioxane (5 ml) anddegassed (sonication for 5 minutes then saturated with N₂). PdCl₂dppf(40 mg, 0.050 mmol) and dppf (27.7 mg, 0.05 mmol) was then added and thereaction mixture was then heated at 100° C. for 24 hrs under a vigorousstirring and a N₂ atmosphere. The solvent was removed in vacuo and theresidue was purified by column chromatography (silica; ethylacetate:ethanol; 9:1) to give an oil which was used without furtherpurification (116 mg, 32%). m/z (LC-MS, ESP): 357 (M⁺+1).

[0290] (d) 2-(1-Fluoro-9-oxo-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (13)

[0291] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (100 mg, 0.46 mmol),1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-thioxanthen-9-one(12) (110 mg, 0.31 mmol), and ground potassium carbonate (63 mg, 0.62mmol) were suspended in dioxane (5 ml) and degassed (sonication for 5minutes then saturated with N₂). Pd(PPh₃)₄ (18 mg, 0.016 mmol) was thenadded and the reaction mixture was then heated at 90° C. for 24 hoursunder a vigorous stirring and a N₂ atmosphere. The solvent were removedin vacuo and the residue was then suspended in water (50 ml) andextracted with ethyl acetate (3×50 ml). The organics were combined,washed with saturated brine and dried over sodium sulphate. The solventwas removed in vacuo and the residue was purified by columnchromatography (silica; ethyl acetate:ethanol; 9:1) to give the titlecompound as a white solid (5 mg, 4%). m/z (LC-MS, ESP): 410 (M⁺+1).

Example 7 Synthesis of2-(1-Fluoro-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (17)

[0292]

[0293] a: BH₃-THF; b: Tf₂O, pyridine; c: bis(pinacolato)diboron,PdCl₂dppf, dppf, dioxane,100° C.; d: chloropyranone, Pd(PPh₃)₄, dioxane,90° C.

[0294] (a) 1-Fluoro-4-hydroxy-thioxanthen-9-one (14)

[0295] 1-Fluoro-4-hydroxy-thioxanthen-9-one (4.93 g, 20 mmol) wasdissolved in THF (50 ml) and cooled down to 0° C. under a N₂ atmosphere.Borane-tetrahydrofuran complex (1M, 60 ml, 60 mmol) was added drop wiseto the stirred solution over 10 minutes. The reaction was left to reactovernight and was then quenched with acetone (100 ml). The mixture wasevaporated to dryness and the residue was taken into water (200 ml). Theproduct was extracted in ethyl acetate (3×100 ml) and the organics werecombined, dried over sodium sulphate and evaporated in vacuo. Theresidue was purified by column chromatography (silica, hexane:ethylacetate, 9:1) to give a white solid which is readily oxidised by air(2.19 g, 47%). ¹H-NMR (300 MHz, DMSO-d₆): δ_(H)=3.86 (2H, s); 6.73 (1H,m); 6.95 (1H, m); 7.24 (2H, m) 7.47 (2H, m); 10.07 (1H, s).

[0296] (b) 1-Fluoro-9H-thioxanthen-4-yl Trifluoromethane Sulfonate (15)

[0297] 1-Fluoro-4-hydroxy-thioxanthen-9-one (1.66 g, 7.15 mmol) wasdissolved in dry pyridine (35 ml) and cooled to 0° C. under a nitrogenatmosphere. Triflic anhydride (2.22 g, 7.87 mmol) was added dropwise tothe stirred solution over 5 minutes. The reaction was left to react for4 hours at room temperature and was then pour onto water (350 ml). Themilky solution was extracted with DCM (3×200 ml), the organics werecombined and dried over magnesium sulphate. The solvent was removed invacuo and the solid obtained was purified through a plug of silica(ethyl acetate:hexane; 3:97) to give the title compound as a whitefluffy solid (2.55 g, 98%). ¹H-NMR (300 MHz, DMSO-d₆): δ_(H)=3.86 (2H,s); 7.3-7.6 (6H, m)

[0298] (c)1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthe(16)

[0299] 1-fluoro-9H-thioxanthen-4-yl trifluoromethane sulfonate (1 g,2.75 mmol), bis(pinacolato)diboron (840 mg, 3.30 mmol), and groundpotassium acetate (809 mg, 8.25 mmol) were suspended in dioxane (7 ml)and degassed (sonication for 5 minutes then saturated with N₂).PdCl₂dppf (0.112 mg, 0.138 mmol) and dppf (77 mg, 0.138 mmol) was thenadded and the reaction mixture was then heated at 100° C. for 24 hoursunder a vigorous stirring and a N₂ atmosphere. The solvent were removedin vaccuo and the residue was purified by column chromatography (silica;ethyl acetate:ethanol; 9:1) to give an oil which was used withoutfurther purification (460 mg, 49%).

[0300] (d) 2-(1-Fluoro-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one(17)

[0301] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(252 mg, 1.17 mmol),1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthe(400 mg, 1.17 mmol), and ground potassium carbonate (239 mg, 2.34 mmol)were suspended in dioxane (7 ml) and degassed (sonication for 5 minutesthen saturated with N₂). Pd(PPh₃)₄ (67 mg, 0.059 mmol) was then addedand the reaction mixture was then heated at 90° C. for 24 hrs under avigorous stirring and a N₂ atmosphere. The solvent were removed invaccuo and the residue was purified by column chromatography (silica;ethyl acetate:ethanol; 9:1) to give an off white solid which wastriturated in ether and gave the title compound as a white solid (72.3mg, 16%). ¹H-NMR (300 MHz, DMSO-d₆): δ_(H)=3.41 (4H, t); 3.71 (4H, t)5.50 (1H, d); 6.21 (1H, d); 7.25-7.35 (3H, m); 7.52-7.62 (3H, m). m/z(LC-MS, ESP): 396 (M⁺+1).

Example 8 Synthesis of 2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4-thione(18)

[0302]

[0303] 2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4-one (4)(140 mg, 0.354mmol) was dissolved in toluene (5 ml). Lawesson's reagent (215 mg, 0.53mmol) was added to the solution and the mixture was refluxed overnightunder nitrogen with stirring. The toluene was evaporated off in vacuoand the residue was purified via column chromatography (silica,dichloromethane) to give the desired compound (18) as dark orange solid(27 mg, 18%).¹H-NMR (300 MHz, DMSO-d₆): δ_(H)=3.56 (4H, t, J 5 Hz); 3.73(4H, t, J 5 Hz); 7.83 (1H, d, J 2 Hz); 7.76 (1H, d, J 2 Hz); 7.30-7.80(7H, m). m/z (LC-MS, ESP):412 (M⁺+1).

Example 9 2-(7-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-oneN-amide Derivatives

[0304]

[0305] 2-(2-Methoxy-phenylsulfanyl)-5-nitro-benzoic Acid

[0306] 2-Methoxythiophenol (9.9 ml, 81.29 mmol) was added to a solutionof KOH (18.24 g, 325.18 mmol) in water (80 ml) degassed for 15 minutes.2-Bromo-5-nitrobenzoic acid (20.0 g, 81.29 mmol) and copper bronze (258mg, 4.06 mmol) were added to the reaction mixture, which was refluxedovernight. The reaction was stopped and the mixture was filtered througha celite pad and washed with 2M NaOH then water (50 ml). The filtratewas acidified (pH 1) with concentrated HCl. The precipitate formed wasfiltered and dried overnight in a vacuum oven (50° C.) to give the crudetitle compound (26.0 g) as a pale yellow solid. The product was usedwithout further purification.

[0307] 5-Methoxy-2-nitro-thioxanthene-9-one

[0308] 2-(2-Methoxy-phenylsulfanyl)-5-nitro-benzoic acid (13.00 g, 42.58mmol) was suspended in methanesulphonic acid (100 ml) and heated at 100°C. The crude mixture was slowly poured onto ice with vigorous stirringthen neutralized with conc. ammonia solution. The precipitate wasfiltered and washed with water. The yellow/lime colored solid was driedunder vacuum at 50° C. to give the crude title compound which was usedwithout any further purification (12 g, 98%). m/z (LC-MS, ESP), RT=4.89min, (M⁺+1)=288.

[0309]5-Methoxy-2-nitro 9H-thioxanthene

[0310] To a cooled (0° C.) suspension of5-methoxy-2-nitro-thioxanthene-9-one (24.46 g, 85.13 mmol) in anhydroustetrahydrofuran (40 ml) under nitrogen atmosphere, was added drop wiseborane-THF complex (170 ml, 1.0M in THF). The mixture was allowed towarm to room temperature with stirring overnight. The reaction mixturewas cooled (0° C.) and the excess borane was quenched with acetone. Thesolvent was evaporated in vacuo. The residue was purified by flashchromatography (1:1, dichloromethane/hexane) to give the title compound(11.59 g, 50%) as a bright yellow amorphous solid. ¹HNMR (300 MHz,DMSO-d₆): δ_(H)=3.86 (3H, s), 4.03 (2H, s), 7.00 (2H, dd), 7.28 (1H, t),7.73 (1H, d), 8.05 (1H, dd), 8.28 (1H, d).

[0311] 5-Methoxy-9H-thioxanthen-2-ylamine

[0312] 5-Methoxy-2-nitro 9H-thioxanthene (11.59 g, 42.40 mmol) wassuspended in ethyl acetate (250 ml). SnCl₂.2H₂O (47.84 g, 212 mmol) wasadded and the clear yellow solution was stirred at 50° C. overnight. Thereaction was the quenched with NaOH (2M) and then extracted with ethylacetate (3×300 ml). The organics were washed with saturated brine (100ml), dried over magnesium sulphate and the solvents were removed invacuo to give the title compound (10.32 g, 100%) as viscous yellow oil.The oil was used without further purification. ¹HNMR (300 MHz, DMSO-d₆):δ_(H)=3.83 (3H, s), 3.67 (2H, s), 5.14 (2H, bs), 6.43 (1H, dd), 6.61(1H, d), 6.89 (1H, d), 6.99 (1H, d), 7.06 (1H, d), 7.18 (1H, t). m/z(LC-MS, ESP), RT=3.88 min, (M⁺+1)=244.

[0313] 7-Amino-9H-thixanthen-4-ol

[0314] 5-Methoxy-9H-thioxanthen-2-ylamine (10.32 g, 41.09 mmol) andpyridine hydrochloride (49.0 g, 424 mmol) were heated at 200° C. undernitrogen atmosphere for 5 hours. The black reaction mixture was allowedto cool down to room temperature and water (50 ml) was then added. Themixture was neutralized with NaOH (2M) to pH 7 then extracted withdichloromethane (4×100 ml). The organics were washed with saturatedbrine, dried over MgSO₄) and concentrated in vacuo to give a black oil.This oil was purified by flash chromatography (dichloromethane) to givethe title compound (7.78 g, 80%) as dark brown oil which was usedwithout further purification. ¹HNMR (300 MHz, DMSO-d₆): δ_(H)=3.61 (2H,s), 5.08 (2H, bs), 6.42 (1H, dd), 6.58 (1H, d), 6.69 (1H, d), 6.81 (1H,d) 6.95-7.06 (2H, m), 9.88 (1H, bs); m/z (LC-MS, ESP), RT═3.23 min,(M⁺+1)=230.

[0315] (5-Hydroxy-9H-thioxanthen-2-yl)-carbamic Acid tert-Butyl Ester

[0316] To a solution of 7-amino-9H-thixanthen-4-ol (7.77 g, 81.32 mmol)in THF (14 ml) was added dropwise di-tert-butyl dicarbonate (17.74 mg,0.49 mmol) in THF (4 ml). The reaction was stirred at room temperatureunder nitrogen atmosphere. Upon completion of the reaction the solventwas evaporated. The residue was taken up in methanol (50 ml), and sodiumhydroxide (4.06 g, 101.16 mmol) was added. The dark brown mixture wasrefluxed for 20 minutes. The solvent was evaporated in vacuo and the oilwas taken up in water, extracted with ethyl acetate, dried over MgSO₄and evaporated in vacuo to give the crude product. The dark brown oilwas purified by flash chromatography (dichloromethane) to give the titlecompound (4.2 g, 38%), as a cream coloured amorphous solid. ¹HNMR (300MHz, DMSO-d₆): δ_(H)=3.74 (2H, s), 6.74 (1H, d), 6.87 (1H, d), 7.04 (1H,t), 7.23-7.33 (2H, m), 7.57 (1H, bs), 10.03 (1H, bs).

[0317] (5-Trifluoromethanesulfonyl-9H-thioxanthen-2-yl)-carbamic Acidtert-Butyl Ester

[0318] To a cooled (0° C.) golden colored solution of(5-hydroxy-9H-thioxanthen-2-yl)-carbamic acid tert-butyl ester (4.0 g,12.14 mmol) in anhydrous pyridine (8 ml) under nitrogen atmosphere wasadded trifluoromethanesulphonic anhydride (2.36 ml, 13.35 mmol) dropwise. The solution turned deep orange upon addition oftrifluoromethanesulphonic anhydride. The reaction was allowed to warm toroom temperature. After 10 minutes of stirring at this temperature thesolution was poured into water (20 ml). The product was extracted withethyl acetate. The organics were washed with saturated brine, dried overMgSO₄ and concentrated in vacuo to give the title compound (5.6 g, 100%)as a dark orange solid.

[0319] [5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-2-yl]-carbamic Acid tert-Butyl Ester

[0320] (5-Trifluoromethanesulfonyl-9H-thioxanthen-2-yl)-carbamic acidtert-butyl ester (3.31 g, 7.17 mmol), bis(pinacolato)diboron (2.18 g,8.6 mmol) and potassium acetate (2.11 g, 21.5 mmol) in 1,4-dioxane (20ml) was degassed for 15 minutes. To the yellow suspension was then addedPdCl₂(dppf) (293 mg, 0.36 mmol) and dppf (199 mg, 0.36 mmol). The darkred mixture was heated to 90° C. under a N₂ atmosphere for 48 hours. Thecrude mixture was purified by flash chromatography (dichloromethane) togive viscous brown oil (3.15 g), which was used without any furtherpurification.

[0321] [5-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl]-carbamic Acid tert-Butyl Ester (19)

[0322][5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-2-yl]-carbamicacid tert-butyl ester (1.02 g, 2.32 mmol),2-chloro-6-morpholin-4-yl-pyran-4-one (3) (0.60 g, 2.78 mmol) and K₂CO₃(0.64 g, 4.64 mmol) were dissolved in dry 1,4-dioxane (ml). The mixturewas degassed for 15 minutes and Pd(PPh₃)₄ (0.13 g, 0.12mol) was thenadded The dark brown mixture was heated to 90° C. under an atmosphere ofN₂ for 24 hour. The reaction mixture was concentrated in vacuo and water(50 ml) was added. The brown solid was filtered and washed with water(1.21 g, 88%). m/z (LC-MS, ESP), RT=4.6 minutes, (M⁺+1)=493.

[0323] 2-(7-Amino-9H-thioxanthen-4-yl) -6-morpholin-4-yl-pyran-4-one(20)

[0324] To a solution of[5-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl]-carbamicacid tert-butyl ester (19)(1.08 g, 2.19 mmol) in dichloromethane (10 ml)was added trifluoroacetic acid (2 ml) and left under stirring at roomtemperature overnight. The solvent was dried in vacuo revealing aviscous dark brown liquid. Saturated sodium bicarbonate solution (20 ml)was added to the residue, which was left to stir for 20 mins. The brownprecipitate was filtered, washing with water and left to dry in thevacuum oven overnight (0.77 g, 90%). ¹HNMR (300 MHz, DMSO-d₆):δ_(H)=3.40 (4H, t), 3.70 (4H, t), 3.77 (2H, s), 5.23 (2H, bs), 5.50 (1H,d), 6.17 (1H, d), 6.44 (1H, dd), 6.65 (1H, d), 7.09 (1H, d), 7.35 (1H,t), 7.47-7.59 (2H, m); m/z (LC-MS, ESP), RT=3.51 minutes, (M⁺+1)=392.

[0325] 2-(7-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-oneN-amide Derivatives

[0326] (a) To a small test tube was added2-(7-amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (20)(20 mg,0.05 mmol), dry dimethylacetamide (0.5 ml), triethylamine (0.01 ml, 0.08mmol) and the desired acid chloride (0.08 mmol) with stirring overnight.The reaction was purified by preparative HPLC to give the desiredproducts, which are shown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 21

90 3.46 435 22

90 3.62 465 23

90 3.58 493 24

95 3.82 461 25

95 3.66 521 26

90 3.53 498 27

90 4.16 487 28

90 3.43 498 29

95 4.44 527 30

90 4.1 503 31

95 4.03 517 32

95 3.99 475 33

90 4.13 516 34

90 3.64 479 35

90 4.12 517 36

90 3.43 546 37

90 3.91 555 38

90 4.16 587 39

90 359 507 40

90 3.5 493 41

90 4.1 569 42

85 4.31 515 43

95 4.16 539 44

95 446 573 45

95 4.02 502 46

95 4.72 586 47

95 3.67 488 48

95 3.42 493 49

95 3.38 505 50

90 3.48 565

[0327] (b) To a small test tube was added2-(7-amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (20)(20 mg,0.05 mmol), dry dimethylacetamide (0.5 ml), triethylamine (8 μl, 0.06mmol) and chloroacetyl chloride (4 μl, 0.06 mmol) with stirringovernight. The appropriate amine or thiol (20 mg or 20 μl) was thenadded and left to stir at room temperature overnight. The reaction waspurified by preparative HPLC to give the desired products, which areshown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 51

90 3.07 518 52

95 2.98 519 53

95 2.98 533 54

95 2.98 538 55

95 3.23 566 56

95 2.93 494 57

95 2.74 493 58

95 3.06 504 59

90 2.99 547 60

95 2.93 533 61

95 3.82 532 62

95 2.77 577 63

95 3.18 570 64

95 3.4 534 65

95 3.1 506 66

95 2.96 450 67

95 2.97 563 68

95 3.16 534

[0328] (c) To a small test tube was added2-(7-amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (20) (20mg, 0.05 mmol), dry dimethylacetamide (0.5 ml), triethylamine (8 μl,0.06 mmol) and 3-bromopropionyl chloride (5 μl, 0.05 mmol) with stirringovernight. The appropriate amine or thiol (2mg or 20 μl, hydrochloridesalts were freed by addition of triethylamine) was then added and leftto stir at room temperature overnight. The reaction was purified bypreparative HPLC to give the desired products, which are shown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 69

90 3.17 532 70

90 2.88 533 71

95 2.98 547 72

95 2.95 552 73

95 3.19 580 74

95 2.95 508 75

90 2.76 507 76

95 3.08 518 77

90 2.81 561 78

95 2.83 547 79

90 3.2 546 80

95 2.84 591 81

95 3.3 584 82

95 3.43 548 83

90 3.06 520 84

90 2.98 464 85

95 2.89 577 86

95 3.14 548

Example 10 2-(4-Hydroxy-9H-thioxanthen-lyl)-6-morpholin-4-yl-pyran-4-oneEther Derivatives

[0329]

[0330] 1-Bromo-4-hydroxy-thioxanthen-9-one

[0331] Thiosalicylic acid (20.0 g, 129.71 mmol) and 4-bromophenol (35.9g, 207.53 mmol) were suspended in conc. H₂SO₄ (200 ml) and stirred for48 hours. The red solution was slowly poured onto ice (500 ml) withvigorous stirring. The resulting yellow precipitate was filtered, anddried in a vacuum oven (50° C.) to give the title compound (24.23 g,61%) as a yellow amorphous solid. m/z (LC-MS, ESP), RT=4.39 min,(M⁻−1)=305-307.

[0332] 1-Bromo-9H-thioxanthen-4-ol

[0333] To a cooled 0° C.) suspension of1-bromo-4-hydroxy-thioxanthen-9-one (24.23 g, 78.88 mmol) in anhydroustetrahydrofuran (40ml) under nitrogen atmosphere, was added dropwiseborane-THF complex (237 ml, 1M in THF). The cloudy mixture was allowedto warm to room temperature and was left stirring overnight. Thesuspension dissolved gradually as the reaction progressed giving ayellow solution. The reaction mixture was cooled (0° C.) and the excessborane was quenched with acetone. The yellow solution was evaporated invacuo. The resulting oil was purified by flash chromatography (4:1,hexane/ethyl acetate) to give the title compound (11.50 g, 50%). m/z(LC-MS, ESP), RT=4.84 min, (M⁻−1)=291-293.

[0334] Carbonic acid tert-Butyl Ester1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-ylEster

[0335] To a stirred solution of 1-bromo-9H-thioxanthen-4-ol (11.50 g,39.22 mmol) in pyridine (7 ml) was added triethylamine (8.15 ml, 58.83mmol). To the solution was added dropwise di-tert-butyl dicarbonate(9.41 g, 3.14 mmol) in pyridine (4 ml). After 1 hour of stirring thecrude reaction mixture was poured into water (100 ml) and extracted withdichloromethane (3×100 ml). The organics were washed with sat. brine (50ml), dried over MgSO₄ and the solvent was evaporated in vacuo to givethe title compound (10.40 g, 67%) as a clear viscous oil. ¹HNMR (300MHz, DMSO-d₆): δ_(H)=1.53 (9H, s), 4.09 (2H, s), 7.15-7.65 (6H, m).

[0336] Carbonic Acid tert-Butyl Ester1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-ylEster

[0337] Carbonic acid tert-butyl ester1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-ylester (5.00 g, 12.71 mmol), anhydrous potassium acetate (3.74 g, 38.13mmol), 1,1′-bis(diphenylphosphino)ferrocene (352 mg, 0.64 mmol) andbis(pinacolato)diboron (3.87 g, 15.25 mmol) were suspended in anhydrousdioxane (8 ml) under nitrogen atmosphere. The mixture was degassed for10 minutes anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (514 mg, 0.64 mmol) was added to the mixture. Thereaction was heated at 90° C. under nitrogen atmosphere for 24 hours.The crude reaction mixture was purified by flash chromatography(dichloromethane), to give the title compound (3.02 g) as a crude brownoil which was used without further purification.

[0338] 2-(4-Hydroxy-9H-thioxanthen-1yl) -6-morpholin-4-yl-pyran-4-one(87)

[0339] Carbonic acid tert-butyl ester1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-ylester (3.00 g, 6.81 mmol), 2-chloro-6-morpholin-4-yl-pyran-4-one (1.22g, 5.67 mmol) and potassium carbonate (2.07 g, 14.98 mmol) weresuspended in anhydrous dioxane (6 ml) under nitrogen atmosphere. Thesolution was degassed for 15 minutes. To the solutiontetrakis(triphenylphosphino) palladium (291 mg, 5% eq.) was added. Themixture was degassed for a further 5 minutes. The reaction was heated at90° C. under nitrogen atmosphere for 24 hours. The solvent wasevaporated in vacuo and the crude mixture was purified by columnchromatography (9:1, ethyl acetate/ethanol), to yield the title compound(421 mg, 16%) as a light yellow amorphous solid. ¹H NMR (300 MHz,DMSO-d6): δ_(H)=3.33 (4H, t), 3.67 (4H, t), 3.88 (2H, s), 5.45 (1H, d),6.05 (1H, d), 6.87 (1H, d), 7.24-7.65 (5H, m), 10.62 (1H, bs); m/z(LC-MS, ESP), RT=3.96 min, (M⁺+1)=394.

[0340] 2-(4-Hydroxy-9H-thioxanthen-1yl)-6-morpholin-4-yl -pyran-4 oneEther Derivatives

[0341] (a) To a mixture of2-(4-hydroxy-9H-thioxanthen-1yl)-6-morpholin-4-yl-pyran-4-one (87)(20mg, 0.05 mmol) and potassium carbonate (16 mg, 0.11 mmol) inN,N-dimethylformamide (0.5 ml) was added dibromoethane (22 μl, 0.25mmol). After 4 hours the appropriate amine or thiol (0.254 mmol, 5 eq)was added to the solution, and the compounds isolated are shown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 88

95 3.26 505 89

95 3.00 506 90

95 3.13 520 91

95 3.03 525 92

95 3.3 553 93

95 3.02 481 94

95 2.76 480 95

85 3.03 511 96

90 3.15 491 97

90 2.88 534 98

90 2.83 520 99

95 3.28 519 100

95 3.08 465 101

95 3.38 557 102

90 3.7 521

[0342] (b) 2-(4-hydroxy-9H-thioxanthen-lyl)-6-morpholin-4-yl-pyran-4-one(87)(20 mg, 0.0508 mmol), potassium carbonate (44 mg, 0.315 mmol) andN,N-dimethylformamide (0.5 ml) was added to 2, 3 or 4-picolyl chloridehydrochloride (0.25 mmol), respectively. The reactions were stirred atroom temperature for 2 hours. The crude reaction mixtures were submittedfor purification by preparative HPLC without any further workup, and thecompounds produced are shown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 103

95 4.07 485 104

90 3.52 485 105

90 3.37 485

Example 11 N-Acyl2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one Derivatives

[0343]

[0344] 1-Fluoro-4-hydroxy-thioxanthen-9-one

[0345] To a solution of 2-thiosalicylic acid (39.32 g, 255 mmol) inconcentrated sulfuric acid (700 ml) was added 4-fluorophenol (32.0 g,280 mmol). The red solution was then stirred at room temperature for 18hours. Upon completion, the mixture was poured directly onto 4 litres ofcrushed ice and the resulting red solid was filtered off, and thensuspended in water (1 L)and treated with ammonia solution until pH 6attained whereupon the precipitate was re-filtered to give the titlecompound as an orange solid (44.48 g, 70.8%) m/z (LC-MS, ESP): 247[M+H]⁺, R/T=3.99 mins

[0346] 4-Benzyloxy-1-fluoro-thioxanthen-9-one

[0347] K₂CO₃ (21.0 g, 150 mmol) was added to a stirred suspension of1-Fluoro-4-hydroxy-thioxanthen-9-one (18.47 g, 75.0 mmol) in methanol(100 mL) followed by benzylbromide (16 mL, 75.0 mmol) which was added inslow stream via syringe. The resulting mixture was then heated to refluxfor 90 minutes and then cooled to room temperature before it was pouredonto cruched ice (0.5 L). The resulting precipitate was filtered off anddried (P₂O₅) to give the title compound as a yellow solid (16.7 g,66.1%) m/z (LC-MS, ESP): 337 [M+H]⁺, R/T=5.22 mins

[0348] 4-Benzyloxy-l-(4-methoxy-benzylamino)-thioxanthen-9-one

[0349] To a solution of 4-methoxybenzyl amine (1.63 g, 11.89 mmol) indry pyridine (10 ml) was added 4-Benzyloxy-1-fluoro-thioxanthen-9-one (1g, 2.97 mmol) in a single portion. The mixture was then heated to reflux(140° C.) for 18 hrs. The resulting hot orange suspension was allowed tocool to room temperature before being poured onto 100 ml of crushed ice.The precipitate was filtered off and washed with copious amounts ofwater to give the title compound as a red/orange solid (1.35 g, 89.6%).m/z (LC-MS, ESP): 454 [M +H]⁺ R/T=6.09 mins.

[0350] (4-Benzyloxy-9H-thioxanthen-1-yl) -(4-methoxy-benzyl) -amine

[0351] To a cooled (0° C.) suspension of4-benzyloxy-1-(4-methoxy-benzylamino)-thioxanthen-9-one (8.16 g, 18.00mmol) in dry THF (150 ml) was added Borane-THF complex (90 mmol, 90 ml1M in THF) in a dropwise fashion. The reaction was allowed to slowlywarm to room temperature and stirred for a further 16 hours to give ahomogeneous yellow solution. To mixture was then cooled (0° C.) anddiluted slowly with acetone (150 ml) and then stirred for 60 minutes atroom temperature. The solvent was removed in vacuo to give a cruderesidue that was diluted in CH₂Cl₂ (100 ml) and the washed with asaturated solution of NaHCO₃ (100 ml), dried using MgSO4, filtered andconcentrated in vacuo to give the title compound as a mild amber oil(7.90 g, 99.8%) m/z (LC-MS, ESP): 438 [M+H]⁺, R/T=5.01 mins.

[0352] 1-Amino-9H-thioxanthen-4-ol

[0353] (4-Benzyloxy-9H-thioxanthen-1-yl)-(4-methoxy-benzyl)-amine (14.51g, 33.00 mmol) was mixed thoroughly with solid pyridine hydrochloride(190 g, 165.00 mmol) before being heated to 150° C. and stirred at thistemperature for a further 12 hours. Upon completion the reaction wascooled slightly before being poured into an beaker of ice/water. Thebrown precipitate was removed by filtration and the filtrate adjusted topH 11 with NH₃0H solution before being extracted with CH₂Cl₂ (3×100 ml).The combined organic phases were then washed with water (1×100 ml) andbrine (1×100 ml) then dried using MgSO4, filtered and concentrated invacuo to give the title compound as a thick brown oil (7.50 g, 99.1%)m/z (LC-MS, ESP): 229 [M+H]⁺, R/T=4.15 mins.

[0354] (4-Hydroxy-9H-thioxanthen-1-yl)-carbamic Acid tert-Butyl Ester

[0355] To a solution of 1-amino-9H-thioxanthen-4-ol (7.57 g, 33.00 mmol)in dry THF (50 ml) was added di-tertiary butyl dicarbonate (20 g, 91.64mmol) in a single portion. The reaction was stirred at room temperaturefor 4 hours before the addition of methanol (50 mL) and solid NaOH (10g, 250 mmol). The resulting slurry was stirred at room temperature for 1hr before the addition of H₂O (250 ml) and EtOAc (250 ml). The organicextract was removed and the remaining aqueous extracted further withEtOAc (2×50 ml). The combined organics were then dried using MgSO4,filtered and concentrated in vacuo to give the title compound a darkamber oil (10.87 g, 92%) m/z (LC-MS, ESP): 328 [M−H]⁻, R/T=4.73 mins

[0356] Trifluoro-methanesulfonic Acid1-tert-Butoxycarbonylamino-9H-thioxanthen-4-yl Ester

[0357] To a cooled (0° C.) solution of(4-Hydroxy-9H-thioxanthen-1-yl)-carbamic acid tert-butyl ester (10.05 g,30.50 mmol) in dry pyridine (70 ml) was added trifluoromethanesulphonicanhydride (8 ml, 48.77 mmol) in a slow stream via syringe over 10 mins.The brown mixture was stirred at 0° C. for a further 30 mins before theaddition of water in a dropwise fashion. The mixture was extracted withEtOAc (3×100 mL), the organic extracts combined, dried using MgSO4,filtered and concentrated in vacuo to give a pale brown oil.Purification of the crude residue was accomplished by flashchromatography (SiO₂) using Hexanes:EtOAc (4:1) to give a mild amber oilthat was purified by flash chromatography (SiO₂) (Hexanes then3:1-Hexanes:EtOAc) to give a mild amber oil (9.42 g, 67.0%) m/z (LC-MS,ESP): 460 [M−H]⁻, R/T=5.52 mins.

[0358][4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-1-yl]-carbamicAcid tert-Butyl Ester

[0359] To a solution of Trifluoro-methanesulfonic acid1-tert-butoxycarbonylamino-9H-thioxanthen-4-yl ester (3.05 g, 6.60 mmol)in dry dioxane (10 ml) was added bis(pinacolato)diboron (2.0 g, 7.92mmol) and anhydrous potassium acetate (1.9 g, 19.80 mmol). The reactionwas then degassed (sonication for 20 min then saturated with N₂) beforethe addition of dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (0.26 g). The reaction mixture wasdegassed for a further 20 minutes before a reflux condenser was attachedto the reaction vessel which was then heated to 100° C. and stirredvigorously for 24 hours. The brown reaction mixture was then poured ontoa silica pad prepared in hexanes and eluted with CH₂Cl₂:Hexanes (1:1).The collected eluent was concentrated in vacuo to give crude titlecompound as a dark brown oil that was used without further purification(2.90 g,).

[0360][4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-1-yl]-carbamicAcid tert-Butyl Ester

[0361] [4-(4,4,5, 5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-1-yl]-carbamic acid tert-butyl ester(2.90 g, 6.50 mmol) was introduced to a solution of2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(1.4 g, 6.50 mmol) in anhydrousdioxane (6 mL). Powdered K₂CO₃ (2.01 g, 14.50 mmol) was added and themixture degassed (sonication for 20 mins then saturated with N₂). To thedegassed solution was added Tetrakis (triphenylphosphine) palladium(0.39 g) before it was degassed for a further 20 minutes. A refluxcondenser was attached to the reaction vessel which was submerged intoan oil bath maintained at 100° C. for 14 hours whereupon the goldenmixture was cooled and diluted with EtOAc (50 ml) and then washed withwater (20 ml) and saturated brine (20 ml). Organic extract was driedusing MgSO4, filtered and concentrated in vacuo to give the titlecompound as a light brown oil that was used without furtherpurification. m/z (LC-MS, ESP): 493 [M+H]⁺, R/T=4.41 mins.

[0362] 2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one(106)

[0363] To a solution of[4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-1-yl]-carbamicacid tert-butyl ester (3.25 g) in CH₂Cl₂ (25 ml) was addedtrifluoroacetic acid (5 ml). The mixture was stirred at room temperaturefor 18 hrs wereupon it was cooled (0° C.) and quenched by dropwiseaddition of saturated NaHCO₃ until the pH 9 was attained. The mixturewas then extracted using CH₂Cl₂ (3×20 mL), the combined organic extractswere then dried (MgSO₄), filtered and concentrated in vacuo to give asemi-crystalline solid that was applied onto a thin silica pad andeluted with EtOAc (100%) going to EtOAc:MeOH (9:1). The eluent wasconcentrated in vacuo to give the title compound as a mild amber oil(1.46 g, 56.4% over three steps) m/z (LC-MS, ESP): 393 [M+H]⁺, R/T=3.79mins

[0364] N-Acyl 2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one Derivatives

[0365] (a) To a stirred solution of2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (106)(39mg, 0.1 mmol) in anhydrous N,N-dimethylformamide (1 ml),N-ethyldiisopropylamine (0.4 ml, 2.31 mmol) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(50 mg, 1.3 mmol) were added. The appropriate carboxylic acid (0.1 mmol)was then added and the mixture stirred at room temperature overnight.The compound was then purified by preparative HPLC to give the desiredcompounds, which are shown below:

Compound R Purity M⁺ + 1 107

95 487 108

95 546 109

95 555 110

95 580 111

95 519 112

95 555 113

95 522 114

95 498 115

85 541 116

95 479 117

99 537 118

85 493 119

95 498 120

95 527 121

95 557 122

95 544 123

95 569 124

95 507 125

95 569 126

95 488 127

95 493 128

95 486 129

98 501

[0366] (b) To a solution of2-(1-amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (106)(25mg, 0.06 mmol) and pyridine (0.5 mmol) in CH₂Cl₂ (1 mL) was added theappropriate sulfonyl chloride (0.2 mmol) in a single portion. Thereaction was stirred at room temperature overnight. The resultingreaction mixture was then purified by preparative HPLC to give thedesired compounds, which are shown below:

Compound R Purity M⁺ + 1 130

84 547 131

97 57

Example 122-Morpholin-4-yl-6-(11-oxo-10,11-dihydrodibenzo[b,f]thiepin-4-yl)-pyran-4-one

[0367]

[0368] [2-(2-Methoxy-phenylsulfanyl) -phenyl]-acetic Acid

[0369] 2-Methoxythiophenol (2.8 g, 20 mmol) was added to a solution ofpotassium hydroxide (4.6 g, 80 mmol) in water (50 ml) and the mixturewas degassed for 15 minutes. 2-Iodophenylacetic acid (5.24 g, 20 mmol)and copper bronze (64 mg, 1 mmol) were then added to the reactionmixture, which was refluxed overnight. The solution was cooled down,filtered and the precipitate washed with water (50 ml). The filtrate wasacidified with conc HCl (pH 1), extracted with dichloromethane (3×100ml). The organics were combined, extracted with saturated brine, driedover sodium sulphate and evaporated in vacuo to give the title compoundas a pale brown oil which solidified overnight. The compound was usedwithout any further purification (5.10 g, 93%).

[0370] 6-Methoxy-11H-dibenzo[b,f]thiepin-10-one

[0371] [2-(2-Methoxy-phenylsulfanyl)-phenyl]-acetic acid (5.40 g, 20mmol) was dissolved in methanesulfonic acid (50 ml) and the mixture washeated for 2 hours at 90° C. under stirring and a nitrogen atmosphere.The reaction mixture was cooled to room temperature and poured onto icewith stirring. The black precipitate was filtered and dried over nightin a vacuum oven (50° C.). The compound was used without any furtherpurification (4.60 g, 91%).

[0372] 6-Hydroxy-11H-dibenzo[b,f]thiepin-10-one

[0373] 6-Methoxy-l1H-dibenzo[b,f]thiepin-10-one (1.54 g, 6 mmol) andpyridine hydrochloride (10 g) were heated for 2 hours at 200° C. withstirring and N₂ atmosphere. The reaction was cooled down to roomtemperature and then triturated in water (200 ml) The pale greenprecipitate was filtered and dried overnight in a vacuum oven (50° C.)(1.40 g, 96%). ¹HNMR (300 MHz, DMSO-d₆): δ_(H)=4.20 (2H, s), 7.05-7.69(7H, m), 10.56 (1H, s).

[0374] Trifluoro-methanesulfonic acid11-oxo-10,11-dihydro-dibenzo[b,f]thiepin-4-yl Ester

[0375] 6-Hydroxy-11H-dibenzo[b,f]thiepin-10-one (242 mg, 1 mmol) wasdissolved in dry pyridine (5 ml) and trifluoromethanesuphonic anhydride(0.17 ml, 1 mmol) was added drop wise to the stirred solution at 0° C.under N₂ atmosphere. The reaction mixture was left to react for 4 hrsand was then poured into water. (50 ml) The organic were extracted withdichloromethane (3×50 ml), washed with 0.2N HCl, dried over magnesiumsulphate and evaporated in vaccuo to give a dark brown solid. This solidwas purified by column chromatography (dichloromethane/hexane, 3:7,Rf=0.15) to give the title compound as a pale brown solid (0.37 g,100%). ¹HNMR (300 MHz, DMSO-d₆): δ_(H)=3.70 (2H, s), 7.31-7.8 (7H, m).

[0376]6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-11H-dibenzo[b,f]thiepin-10-one

[0377] Trifluoro-methanesulfonic acid11-oxo-10,11-dihydro-dibenzo[b,f]thiepin-4-yl ester (0.374 g, 1 mmol),bis(pinacolato)diboron (305 mg, 1.2 mmol) and potassium acetate (294 mg,3 mmol) were dissolved in 1,4-dioxane (5 mL) and the mixture wasdegassed for 5 min. Pd(dppf)Cl₂ (40 mg, 0.05 mmol) and dppf (28 mg, 0.05mmol) were added to the vessel, and the reagents heated to 100° C. undernitrogen with stirring for 12 hours. The reaction mixture was purifiedby flash chromatography (dichloromethane/hexane, 1:4) and the blackresidue was used without further purification (0.35 g).

[0378]2-Morpholin-4-yl-6-(11-oxo-10,11-dihydro-dibenzo[b,f]thiepin-4-yl)-pyran-4-one (132)

[0379] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(215 mg, 1 mmol),6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-11H-dibenzo[b,f]thiepin-10-one (352 mg, 1 mmol), and ground potassiumcarbonate (276 mg, 2 mmol) were suspended in 1,4-dioxane (10 ml) anddegassed for 5 minutes. Pd(PPh₃)₄ (57 mg, 0.05 mmol) was then added andthe reaction mixture was then heated at 90° C. for 4hours under avigorous stirring and a N₂ atmosphere. The solvent was removed in vaccuoand the residue was then suspended in water (100 ml). The organics wereextracted with dichioromethane (3×100 ml), combined, washed withsaturated brine and dried over sodium sulphate. The solvent was removedin vaccuo and the residue was purified by column chromatography (silica;ethyl acetate:ethanol; 9:1) to give the title compound as a pale brownsolid (0.12 g, 29%). ¹HNMR (300 MHz, DMSO-d₆): δ_(H)=3.40 (4H, t), 3.70(4H, t), 4.43 (2H, s), 5.55 (1H, d), 6.29 (1H, d), 7.25-7.55 (5H, m),7.78-7.81 (1H, m), 8.20-8.22 (1H, m); m/z (LC-MS, ESP), RT=4.12 min,(M⁺+1)=406.

Example 132-(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-6-morpholin-4-yl-pyran-4-one

[0380]

[0381] 4-Methoxy-10,11-dihydro-dibenzo[b,f]thiepine

[0382] Hydrazine hydrate (4 ml) and potassium hydroxide (2.72 g, 48mmol) was added to 6-methoxy-11H-dibenzo[b,f]thiepin-10-one (4.10 g, 16mmol) in ethylene glycol (20 ml) and the reaction mixture was heated at175° C. for 3 hours. The reaction mixture was cooled down to roomtemperature and water was added (100 ml). The white solution wasextracted with ether (3×200 ml), the organics were combined, washed withwater (100 ml), brine (100 ml) and dried over magnesium sulphate. Thesolvent was removed in vacuo to give an oil which solidify upon standingto give a brown solid which was used without any further purification(2.45 g, 63%).

[0383] 10,11-Dihydro-dibenzo[b,f]thiepin-4-ol

[0384] 4-Methoxy-10,11-dihydro-dibenzo[b,f]thiepine (2.42 g, 10 mmol)and pyridine hydrochloride (15 g) were heated with stirring at 180° C.for one hour. Water (100 ml) was added to the reaction mixture and theorganics were extracted with ethyl acetate (3×100 ml). The organics werecombined and washed with 2N HCl (50 ml), brine (50 ml), and dried overmagnesium sulphate. The solvent were removed in vacuo and the residuewas purified by column chromatography (1:9; dichloromethane:hexane) togive the desired compound as a white solid (1.55 g, 68%). ¹HNMR (300MHz, DMSO-d₆): δ_(H)=3.13-3.23 (4H, m), 6.70 (2H, t), 6.97-7.15 (4H, m),7.38 (1H,s) 9.78 (1H, s); m/z (LC-MS, ESP), RT=4.59 min, (M⁺+1)=229.

[0385] Trifluoro-methanesulfonic acid10,11-dihydro-dibenzo[b,f]thiepin-4-yl Ester

[0386] 10,11-Dihydro-dibenzo[b,f]thiepin-4-ol (1.26 g, 5.5 mmol) wasdissolved in dry pyridine (5 ml) and trifluoromethanesuphonic anhydride(1.12 ml, 6.6 mmol) was added drop wise to the stirred solution at 0° C.under N₂ atmosphere. The reaction mixture was left to react for 4 hrsand was then poured into water. (100 ml) The organic were extracted withdichloromethane (3×50 ml), washed with 0.2N HCl, dried over magnesiumsulphate and evaporated in vaccuo to give a dark brown solid. This solidwas purified by flash chromatography (dichloromethane) to give an oil(1.1 g, 56%). ¹HNMR (300 MHz, DMSO-d₆): δ_(H)=3.25-3.29 (2H, m),3.37-3.41 (2H, m), 7.12-7.17 (1H, m), 7.21-7.31 (3H, m), 7.38-7.41 (3H,s).

[0387]2-(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane

[0388] Trifluoro-methanesulfonic acid10,11-dihydro-dibenzo[b,f]thiepin-4-yl ester (1.08 g, 3 mmol),bis(pinacolato)diboron (914 mg, 3.6 mmol) and potassium acetate (883 mg,9 mmol) were dissolved in 1,4-dioxane (10 mL) and the mixture wasdegassed for 5 minutes. Pd(dppf)Cl₂ (121 mg, 0.15 mmol) and dppf (83 mg,0.15 mmol) were added to the vessel, and the reagents heated to 100° C.under nitrogen with stirring for 12-hours. The reaction mixture waspurified by flash chromatography (dichloromethane) and the black residuewas used without further purification (0.87 g).

[0389] 2-(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-6-morpholin-4-yl-pyran-4-one

[0390] 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(1.12 g, 5.2 mmol),2-(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane(880 mg, 2.6 mmol), and ground potassium carbonate (720 mg, 5.2 mmol)were suspended in 1,4-dioxane (10 ml) and degassed for 5 minutes.bis(tri-t-butylphosphine)palladium (66 mg, 0.13 mmol) was then added andthe reaction mixture was then heated at 90° C. for 4 hours under avigorous stirring and a N₂ atmosphere. The solvent was removed in vaccuoand the residue was then suspended in water (100 ml). The organics wereextracted with dichloromethane (3×100 ml), combined, washed withsaturated brine and dried over sodium sulphate. The solvent was removedin vaccuo and the residue was purified by column chromatography (silica;ethyl acetate:ethanol; 9:1) to give a pale brown solid (50 mg, 5%).¹HNMR (300 MHz, DMSO-d₆): δ_(H)=3.24-3.32 (6H, m), 3.44 (2H, t), 3.66(4H, t), 5.50 (1H, d), 6.10 (1H, d), 7.08-7.51 (7H, m); m/z (LC-MS,ESP), RT=4.48 min, (M⁺+1)=392.

Example 14 2-Morpholin-4-yl-6-(10H-phenothiazin-4-yl)-pyran-4-one

[0391]

[0392] 10H-Phenothiazin-4-ol

[0393] To a solution of 3-phenylamino-phenol (5 g, 26.99 mmol) in1,2-dichlorobenzene (50 ml) was added S₈ sulfur(1.82 g, 56.76 mmol) in asingle portion and iodine (0.1 g, 0.39 mmol) which was added in threeportions over 10 minutes. A reflux condenser was attached to thereaction vessel which was heated to 185° C. under a nitrogen atmosphere.The mixture was stirred at this temperature for 4 hours and then allowedto cool to room temperature. The reaction mixture was filtered to removea black precipitate and the filtrate diluted with Et₂O (100 ml) andwashed with water (2×100 ml). The organic layer was separated and thevolatile solvents removed to give a deep green oil that was purified byflash column chromatography (SiO₂) (Hexanes then 8:1-Hexanes:EtOAc) togive a pale yellow solid (2.38 g, 40.96%) m/z (LC-MS, ESP) 216 [M+H]⁺,R/T=4.12 mins.

[0394] 4-Hydroxy-phenothiazine-10-carboxylic Acid tert-Butyl Ester

[0395] To a solution of 10H-Phenothiazin-4-ol (0.77 g, 3.58 mmol) inanhydrous pyridine (10 ml) was added di-tertiary butyl dicarbonate (3.12g, 14.31 mmol) in a single portion. The solution was heated to 80° C.and stirred under a nitrogen atmosphere for 60 minutes before beingallowed to cool to room temperature and treated with water (20 ml) andextracted with EtOAc (2×30 ml). The organic layers were then washed withwater (20 ml), dried using MgSO₄, filrtered and concentrated in vacuo togive an amber oil. The crude residue was treatyed with MeOH (15 ml) andsolid NaOH (0.65 g, 16.25 mmol). The mixture was heated to 80° C. for 60minutes then cooled to room temperature and neutralised to pH7 with 1MHCl solution. The resulting suspension was then filtered and dried togive the title compound as a beige solid (1.13 g, 100%) that was usedwithout further purification. m/z (LC-MS, ESP): 315 [M−H]⁻, R/T=4.72mins.

[0396] 4-Trifluoromethanesulfonyloxy-phenothiazine-10-carboxylic Acidtert-Butyl Ester

[0397] Trifluoromethanesulfonic anhydride (2.95 ml, 17.09 mmol) wasadded in a dropwise fashion over 10 minutes to a cooled (0° C.) stirredsolution of 4-Hydroxy-phenothiazine-10-carboxylic acid tert-butyl ester(3.60 g, 11.41 mmol) in pyridine (40 ml). The reaction mixture wasstirred at 0° C. for 1 hour before the addition of water (80 ml). Themixture was extracted using EtOAc (2x60 ml). The organic extracts werethen dried using MgSO₄, filtered and concentrated in vacuo to give adark brown oil. The crude residue was then purified by flashchromatography (SiO₂) (4:1-Hexanes:EtOAc) to yield a yellow oil (5.02 g,98.24%) m/z (LC-MS, ESP): 348 [M+H-BOC]⁺, R/T=5.61 mins

[0398] 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenothiazine-10-carboxylic Acid tert-Butyl Ester

[0399] To a stirred solution of4-trifluoromethanesulfonyloxy-phenothiazine-10-carboxylic acidtert-butyl ester (3.0 g, 6.7 mmol) in anhydrous dioxane (10 ml) wasadded bis(pinacolato)diboron (2.05 g, 8.06 mmol) and potassium acetate(1.96 g, 20.01 mmol). The reaction was then degassed (sonication for 20minutes then saturated with N₂) before the addition ofdichloro[1,1′-bis(diphenylphosphino)ferrocene] palladium(II)dichloromethane adduct (0.27 g, 0.33 mmol). The reaction mixture wasdegassed for a further 20 minutess before a reflux condenser wasattached to the reaction vessel which was then heated to 90° C. andstirred vigorously for 72 hours. The dark brown reaction mixture wasthen allowed to cool to room temperature before it was applied to athick silica pad prepared in hexanes and eluted with hexanes:CH₂Cl₂-(2:1). The eluent was concentrated in vacuo to give a dark brownoil (2.85 g, 100%) that was used for the next transformation with nofurther purification. m/z (LC-MS, ESP): 326 [M+H-BOC]⁺, R/T=5.86 mins

[0400]4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-phenothiazine-10-carboxylicAcid tert-Butyl Ester (134)

[0401] Powdered potassium carbonate (2.03 g, 14.68 mmol) and2-Chloro-6-morpholin-4-yl-pyran-4-one (1.44 g, 6.70 mmol) were added toa stirred solution of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenothiazine-10-carboxylicacid tert-butyl ester (2.85 g, 6.70 mmol) in anhydrous dioxane (20 ml)and the mixture degassed (sonication for 20 minutes then saturated withN₂) thoroughly. Tetrakis (triphenylphosphine) palladium was then addedin a single portion and the mixture degassed (sonication for 20 minutesthen saturated with N₂) once again before a reflux condenser wasattached and the mixture heated to 100° C. under a nitrogen atmospherefor 20 hours. Water (30 ml) was added and the mixture extracted withEtOAc (3×30 ml). The organic extracts were then dried using MgSO₄,filtered and concentrated in vacuo to yield a dark brown, crystallinesolid (3.21 g, 100%) that was taken forward with no furtherpurification. m/z (LC-MS, ESP): 479 [M+H]⁺, R/T=4.55 mins

[0402] 2-Morpholin-4-yl-6-(10H-phenothiazin-4-yl)-pyran-4-one (135)

[0403] To a stirred solution of4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-phenothiazine-10-carboxylicacid tert-butyl ester (3.65 g, 7.63 mmol), in CH₂Cl₂ (30 ml) was addedtrifluoroacetic acid in a single portion. The mixture was stirred atroom temperature for 20 hours whereupon the reaction was concentrated invacuo to give a thick syrup that was basified in a dropwise fashion withsaturated NaHCO₃ (40 ml). The dark green mixture was then stirred atroom temperature for 18 hours. The mixture was filtered and the filtrantretained, washed with water and dried to give the title compound as adark green solid (2.89 g, 83.74% over 3 steps) m/z (LC-MS, ESP): 479[M+H]⁺, R/T=4.05 mins

Example 15 4-Morpholin-4-yl-6-thianthren-1-yl-pyran-2-one

[0404]

[0405] Thianthrene-1-carboxylic Acid

[0406] t-Butyl lithium (1.7M in hexane, 55.1 ml, 93.6 mmol) was addeddrop wise to a stirred solution of thianthrene (16.9 g, 78 mmol) in dryTHF (250 ml) at −78° C. over 30 minutes under an inert atmosphere (N₂).The reaction mixture was allowed to warm to room temperature and theresulting reddish solution was left stirring for 24 hours. The mixturewas then cooled down to −78° C. and carbon dioxide (from dry ice pelletand dried by passing over some activated A4 sieves) was bubbled into thesolution for 1 hour. The reaction was warmed up back to room temperaturewith CO₂ still bubbling through it for another hour. Water (10 ml) wasthen added carefully to the solution and the pH was adjusted to 1 (pHpaper) with 2N HCl. The solvent was removed in vacuo and the yellowsolid formed was filtered and dried overnight in a vacuum desiccators.The solid was then recrystalised from methanol to give the desiredproduct as a pale yellow crystalline solid (11.9 g, 59%). ¹HNMR (300MHz, CDCl₃): δ_(H)=7.25 (3H, m); 7.50 (4H, m). m/z (LC-MS, ESP): RT=4.53min, (M⁻−1)=259

[0407] 1-Thianthren-1-yl-ethanone

[0408] Methyl lithium (1.6M in ether, 57 ml, 90 mmol) was added dropwise to a stirred solution of thianthrene-l-carboxylic acid (11.71 g, 45mmol) in dry tetrahydrofuran (200 ml) at −78° C. over 30 minutes underan inert atmosphere (N₂). The reaction mixture was allowed to warm toroom temperature and (very thick white suspension present) was leftstirring for 4 hours. Water (10 ml) was then added carefully to thesolution and the pH was adjusted to 1 (pH paper) with 2N HCl. Thesolvent was removed in vacuo and the yellow solid formed was filteredand dried overnight in a vacuum desiccators. The solid was then purifiedby column chromatography (ethyl acetate/hexane; 1:9) and wasrecrystalised from ethanol to give the desired product (6.58 g, 57%).¹HNMR (300 MHz, CDCl₃): δ_(H)=2.65 (3H, s); 7.26 (3H, m); 7.47 (2H, m);7.62 (2H, d). m/z (LC-MS, ESP) RT=4.95 min; (M⁺+1)=259

[0409] 3-Oxo-3-thianthren-1-yl-dithiopropionic Acid

[0410] A solution of CS₂ (1.55 ml, 25.5 mmol) and1-thianthren-1-yl-ethanone (6.59 g, 25.5 mmol) in dry tetrahydrofuran(20 ml) was added drop wise to a solution of potassium t-butoxide (5.73g, 51 mmol) in dry tetrahydrofuran (50 ml) under N₂ at 0° C. A redcoloration and the formation of a precipitate were observed. The mixturewas left under vigorous stirring over the weekend and was then pouredonto water (200 ml) and extracted with ether (3×100 ml). The aqueous wasacidified with 2N H₂SO₄ to pH 1 (Whatmann pH paper) and the extractedwith ether (3×100 ml). The organic were dried over magnesium sulphateand the solvent was evaporated in vacuo to give the desired product as adark orange resin (5.00 g, 59%). m/z (LC-MS, ESP), RT=5.11; (M⁻−1)=333

[0411] 3-oxo-3-Thianthren-1-yl-dithiopropionic Acid Ethyl Ester

[0412] Tetrabutylammonium hydrogen sulphate (5.1 g, 15 mmol) and sodiumhydroxide (1.2 g, 30 mmol) were dissolved in water (50 ml) A solution of3-oxo-3-thianthren-1-yl-dithiopropionic acid (5.02 g, 15 mmol) indichloromethane (50 ml) was added to the solution in one portion and wasstirred vigorously for 30 minutes. The aqueous layer was removed andiodoethane (4 ml) was added to the dichloromethane solution that wasthen stirred for 1 hr. The solvent was removed in vacuo and the residuewas taken into water (200 ml) The organics were extracted with ether(3×100 ml), dried over magnesium sulphate and evaporated in vacuo. Theresidue was then purified by column chromatography (ethylacetate:hexane; 1:4) to give the desired compound as a bright yellowsolid (4.00 g, 73%). ¹HNMR (300 MHz, CDCl₃): δ_(H)=1.43 (3H, t), 3.33(3H, q), 6.57 (1H, s), 7.26 (3H, m), 7.51 (3H, m), 7.60 (1H, m), 15.09(1H, s); m/z (LC-MS, ESP), RT=6.50 min, (M⁻−1)=361.

[0413] 3-Morpholin-4-yl-1-thianthren-1-yl-3-thioxo-propan-1-one

[0414] Morpholine (0.96 ml, 11 mmol) was added to a solution of3-oxo-3-thianthren-1-yl-dithiopropionic acid ethyl ester (3.99 g, 11mmol) in ethanol (20 ml). The reaction was refluxed for 8 hours and wasthen cooled to room temperature. The precipitate formed was filtered anddried to give the desired product as a bright orange solid (3.50 g,82%). m/z (LC-MS, ESP), RT=4.81and 5.33min same (M+1)=388

[0415] 3-Ethylsulfanyl-3-morpholin-4-yl-1-thianthren-1-yl-propenone

[0416] 3-Morpholin-4-yl-1-thianthren-1-yl-3-thioxo-propan-1-one (3.49 g,9 mmol), iodoethane. (0.8 ml, 10 mmol), and grinded potassium carbonate(1.38 g, 10 mmol) were suspended in acetone (20 ml) and the mixture wasrefluxed for 24 hours. The solvent was removed in vacuo and the residuewas taken into water (50 ml). The organics were extracted intodichloromethane (3×100 ml), dried over magnesium sulphate and evaporatedin vacuo. The crude product was purified by column chromatography (ethylacetate/hexane) to give the desired product as a yellow solid (2.26 g,60%). ¹HNMR (300 MHz, CDCl₃) δ_(H)=1.34 (3H, t), 2.96 (2H, q), 3.73 (4H,m), 3.84 (4H, m), 7.21 (3H, m), 7.47 (4H, m); m/z (LC-MS, ESP), RT=5.01min, (M⁺+1)=416.

[0417] 4-Morpholin-4-yl-6-thianthren-1-yl-pyran-2-one (136)

[0418] A suspension of activated zinc dust (0.65 g, 10 mmol), ethylbromoacetate (0.56 ml, 5 mmol) and a few crystals of iodine in drytetrahydrofuran (20 ml) were heated at 50° C. for one hour with stirringunder a N₂ atmosphere. A solution of3-ethylsulfanyl-3-morpholin-4-yl-1-thianthren-1-yl-propenone (1.04 g,2.5 mmol) in dry tetrahydrofuran (20 ml) was added drop wise withstirring and the mixture was refluxed for 12 hours under a N₂atmosphere. The mixture was then poured over ice cold dilute 3% H₂SO₄(50 ml), the aqueous layer was extracted with ethyl acetate (3×50 ml),the combined extracts were dried over magnesium sulphate and the solventwas evaporated in vacuo. The residue was purified by columnchromatography (ethyl acetate/hexane) to give the desired product (0.35g, 35%). ¹HNMR (300 MHz, CDCl₃): δ_(H)=3.45 (4H,t), 3.85 (4H, t), 5.35(1H, d), 6.29 (1H, d), 7.26 (3H, m), 7.50 (3H, m) 7.61 (1H, m); m/z(LC-MS, ESP), RT=4.50min, (M⁺+1)=396.

Example 166-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylic AcidAmide Derivatives

[0419]

[0420] 3-Chlorosulfonyl-4-fluoro-benzoic Acid

[0421] Chlorosulphonic acid (100 ml, 1.5 mol) was gradually added to4-fluorobenzoic acid (43 g, 0.307 mol) with stirring. The clear darkyellow mixture was heated to 150° C. for 24 hours. The yellow solutionwas cooled back to room temperature and poured onto ice with vigorousstirring. The white precipitate was filtered and pressed dry. The solidwas dried overnight in a desiccator under vacuum and over activatedsilica (54.65 g, 75%). Mp: 116-117° C.; m/z (LC-MS, ESP), RT=4.03min,(M⁻−1)=237-239 (ratio 1:3).

[0422] 4-Fluoro-3-sulfino-benzoic Acid

[0423] Sodium sulphite (130 g, 1.034 mol) was added slowly to a solutionof 3-chlorosulfonyl-4-fluoro-benzoic acid (49.39 g, 0.207 mol) in water(150 ml) at 0° C. with a vigorous stirring.

[0424] After the addition was completed the reaction was warmed back toroom temperature for 1 hour and the pH of the solution was kept aroundpH 6-7 with 2N sodium hydroxide solution. The white milky suspension wasfiltered and the solid washed with 2N sodium hydroxide solution (150 ml)and then water (100 ml). The filtrate was then cooled in an ice bath andconcentrated HCl was added until no more precipitate was formed (pH<1).The white precipitate was then filtered, pressed dry and left in adessicator overnight under vacuum and over activated silica (27.92 g,66%). m/z (LC-MS, ESP), RT=0.98min, (M⁻−1)=203

[0425] 4-(2-Bromo-phenylsulfanyl)-3-sulfino-benzoic Acid

[0426] 2-Bromobenzenethiol (25 g, 132 mmol) was added to a solution of4-fluoro-3-sulfino-benzoic acid (13.5 g, 66 mmol) and NaOH pellets (11g, 264 mmol) in water (30 ml). The yellow mixture was then degassed for10 minutes and then heated to 140° C. for 48 hours. The reaction wasthen cooled to 0° C. and acidified to pH 4-5 (pH paper) withconcentrated HCl. The precipitate formed was filtered, washed withhexane and was dried in a vacuum dessicator over activated silicaovernight (20.69 g, 84%). m/z (LC-MS, ESP), RT=3.67min, (M⁻−1)=373.

[0427] 6-Bromo-thianthrene-2-carboxylic Acid

[0428] 4-(2-bromo-phenylsulfanyl)-3-sulfino-benzoic acid (14 g, 38 mmol)was added slowly to a stirred solution of methanesulphonic acid (160ml). The purple solution was heated to 60° C. for 3 hours. The reactionwas cooled down to room temperature and was poured into ice (300 ml)where an off-white precipitate appeared. The solid was filtered andwashed with water (100 ml) and then dried in a vacuum dessicator overactivated silica (9.48 g, 73%). ¹HNMR (300 MHz, CDCl₃): δ_(H)=7.29 (1H,t), 7.59 (1H, dd), 7.70 (1H,dd) 7.74 (1H, d), 7.87 (1H, dd), 8.03 (1H,d).m/z (LC-MS, ESP), RT=4.99min, (M⁻−1)=339

[0429] 6-Bromo-thianthrene-2-carboxylic Acid Methyl Ester

[0430] To 6-bromo-thianthrene-2-carboxylic acid (9 g, 28 mmol) inmethanol (180 ml) was slowly added conc. H₂SO₄ (5 ml). The milky whitesuspension was heated to 80° C. until all the solid had gone intosolution (2 hrs). The suspension was concentrated in vacuo. Water (100ml) was added and the organics were then extracted with dichloromethane(3×70 ml), dried over MgSO4 and evaporated in vacuo, yielding to ayellow solid. (4.48 g, 45%). ¹HNMR (300 MHz, CDCl₃): δ_(H)=3.94 (3H, s);7.13 (1H, t), 7.44 (1H, dd), 7.54 (1H,dd) 7.61 (1H, d), 7.93 (1H, dd),8.13 (1H, d).

[0431]6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thianthrene-2-carboxylicAcid Methyl Ester

[0432] 6-Bromo-thianthrene-2-carboxylic acid methyl ester (1 g, 2.8mmol), bis(pinacolato)diboron (0.86 g, 3.4 mmol) and potassium acetate(0.12 g, 0.14 mmol) in 1,4-dioxane (15 ml) was degassed for 15 minutes.To the yellow suspension was then added PdCl₂(dppf) (78 mg, 0.14 mmol)and dppf (0.83 g, 8.5 mmol). The dark red mixture was heated to 90° C.under a N₂ atmosphere for 48 hours. The crude mixture was purified byflash chromatography (dichloromethane) to give viscous brown oil (1.13g), which was used without any further purification.

[0433] 6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylicAcid Methyl Ester (137)

[0434]6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thianthrene-2-carboxylicacid methyl ester (1.1 g, 2.83 mmol),2-chloro-6-morpholin-4-yl-pyran-4-one (0.73 g, 3.4 mmol) and K₂CO₃ (0.8g, 5.66 mmol) were dissolved in dry 1,4-dioxane (7 ml). The mixture wasdegassed for 15 mins and Pd(PPh₃)₄ (0.16 g, 5 mol %) was then added Thedark brown mixture was heated to 90° C. under an atmosphere of N₂ for 24hour. The reaction mixture was concentrated in vacuo and water (100 ml)was added. The brown solid was filtered and washed with water (1.23 g,96%). m/z (LC-MS, ESP), RT=4.49 min, (M⁺+1)=454.

[0435]6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylateSodium Salt (138)

[0436] 6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylicacid methyl ester (1.1 g, 2.43 mmol) and NaOH Pellets (97 mg, 2.43 mmol)were dissolved in methanol (40 ml). The brown suspension was heated to80° C. under N₂ for 24 hours. The solvent was removed in vacuo and theresidue was triturated with diethyl ether. The product was collected byfiltration as a fine dark brown powder (1.11 g, 99%). m/z (LC-MS, ESP),RT=3.90 min, (M⁻−1)=438

[0437] 6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylicAcid Amide Derivatives

[0438]6-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-carboxylatesodium salt (138)(20 mg, 0.04 mmol), HBTU (18 mg, 0.05 mmol),di-isopropylethylamine (9 μl, 0.05 mmol), the appropriate amine (0.04mmol) and dry dimethylacetamide (0.5 ml). The dark brown mixture wasstirred at room temp for 2 hours and then purified by preparative HPLCto give the desired products, which are shown below:

Retention Compound R Purity Time (Mins) M⁺ + 1 139

95 3.65 453 140

95 3.7 467 141

95 4.11 495 142

95 3.78 467 143

95 4.28 495 144

95 4.03 481 145

90 4.22 495 146

95 4.01 481 147

85 3.84 477 148

90 4.1 521 149

90 3.81 523 150

95 4.1 519 151

95 4.54 521 152

95 4.1 493 153

95 4.12 493 154

95 4.77 535 155

95 4.3 507 156

95 3.89 493 157

95 4.14 507 158

95 4.00 537 159

95 4.02 537 160

95 3.69 509 161

95 4.31 521 162

95 3.73 558 163

95 3.72 552 164

95 3.12 537 165

95 3.49 524 166

95 3.29 550 167

95 3.23 536 168

95 3.22 550 169

95 3.62 572 170

95 3.43 530 171

95 3.27 530 172

95 3.21 530 173

95 3.26 544 174

95 3.3 558 175

95 3.05 579 176

95 3.17 533 177

95 3.19 552 178

95 3.18 566 179

95 3.44 641 180

95 3.49 651 181

95 3.29 538 182

95 3.27 552

B) Biological Examples Materials and Methods

[0439] In vitro ATM inhibition Assays

[0440] In order to assess the inhibitory action of the compounds againstATM in vitro, the following assay was used to determine IC₅₀ values.

[0441] ATM protein was immunoprecipitated from HeLa cell nuclear extractusing rabbit polyclonal anti-sera raised to the C-terminal ˜500amino-acid residues of the human ATM protein. The immunoprecipitationwas performed according to the methodology described by Banin, S. et al.(1998). 10 μl of immunoprecipitated ATM in Buffer C (50 mM Hepes, pH7.4, 6 mM MgCl₂, 150 mM NaCl, 0.1 mM sodium orthovanadate, 4 mM MnCl2,0.1 mM dithiothreitol, 10% glycerol) was added to 32.5 μl of buffer Ccontaining 1 μg of the ATM substrate GSTp53N66 in a V-bottomed 96 wellpolypropylene plate. The GSTp53N66 substrate is the amino terminal 66amino acid residues of human wild type p53 fused to glutathioneS-transferase. ATM phosphorylates p53 on the residue serine 15 (Banin,S. et al. (1998)). Varying concentrations of inhibitor were then added.All compounds were diluted in DMSO to give a final assay concentrationof between 100 μM and 1 nM, with DMSO being at a final concentration of1%. After 10 minutes of incubation at 37° C., the reactions wereinitiated by the addition of 5 μl of 500 μM Na-ATP. After 1 hour withshaking at 37° C., 150 μl of phosphate buffered saline (PBS) was addedto the reaction and the plate centrifuged at 1500 rpm for 10 minutes. 5μl of the reaction was then transferred to a 96 well opaque white platecontaining 45 μl of PBS to allow the GSTp53N66 substrate to bind to theplate wells. The plate was covered and incubated at room temperature for1 hour with shaking before discarding the contents. The plate wells werewashed twice by the addition of PBS prior to the addition of 3% (w/v)bovine serum albumin (BSA) in PBS. The plate was incubated at roomtemperature for 1 hour with shaking before discarding the contents andwashing twice with PBS. To the wells, 50 μl of a 1:10,000 dilution ofprimary phosphoserine-15 antibody (Cell Signaling Technology, #9284L) in3% BSA/PBS was added to detect the phosphorylation event on the serine15 residue of p53 elicited by the ATM kinase. After 1 hour of incubationat room temperature with shaking, the wells were washed four times withPBS prior to the addition of an anti-rabbit HRP conjugated secondaryantibody (Pierce, 31462) with shaking for 1 hour at room temperature.The wells were then washed four times with PBS before the addition ofchemiluminescence reagent (NEN Renaissance, NEL105). The plate was thenshaken briefly, covered with a transparent plate seal and transferred toa TopCount NXT for chemiluminescent counting. Counts per second,following a one second counting time, were recorded for each reaction.

[0442] The enzyme activity for each compound is then calculated usingthe following equation:${\% \quad {Inhibition}} = {100 - \left( \frac{\left( {{{cpm}\quad {of}\quad {unknown}} - {{mean}\quad {negative}\quad {cpm}}} \right) \times 100}{\left( {{{mean}\quad {positive}\quad {cpm}} - {{mean}\quad {negative}\quad {cpm}}} \right)} \right)}$

[0443] Sensitisation of Cells to Ionising Radiation or DNA Double StrandBreak Chemotherapies

[0444] To test the efficacy of the ATM inhibitor compound 4 on itsability to sensitise cells to ionising radiation or to DNA double strandbreak inducing chemotherapeutics, clonogenic survival assays wereperformed using the HeLa or LoVo human tumour derived cell lines. TheHeLa line was used for ionising radiation studies while LoVo was usedfor studies with chemotherapeutic agents. Enough cells to give ˜100colonies per treatment were seeded into 6 well dishes 4-6 hours prior tothe addition of compound 4 at the concentrations shown on the graphs.After 1 hour, a concentration range of either etoposide (FIG. 2),camptothecin (FIG. 3) or doxorubicin (FIG. 4) was added. For ionisingradiation treatment (FIG. 1), after 1 hour of incubation with compound4, cells were irradiated at 1 Gy/min using a Faxitron 43855D X-raycabinet. For all treatments, after a further 16 hours incubation, drugcontaining media was removed and fresh media added prior to a furtherincubation of 10 days before the staining of colonies with Giemsa. Allcompounds were solubilised in DMSO, with a final concentration on cellsof no more than 0.1%. Resulting colonies containing >50 cells werecounted as positives.

[0445] Recombinant Retroviral Vectors and Virus Preparation.

[0446] The Ψ⁻/LTR⁻/Vpr⁻ replication deficient HIV-1 gag/pol expressingpackaging constructs were designed based on the vector LΔP2GPH(Haselhorst et al., 1998 Development of cell lines stably expressinghuman immunodeficiency virus type 1 proteins for studies inencapsidation and gene transfer. J Gen Virol, 79, 231-7.). A HIV-1integrase mutant packaging construct, which codes for a D64V amino acidchange in the integrase gene, was made by site directed mutagenesis(Quikchange mutagenesis system, Stratagene). The HIV-1 luciferasetransfer vector, HIV-Luc, was constructed by inserting the fireflyluciferase gene in-between two HIV-1 LTR sequences and a Ψ HIV-1 RNApackaging signal sequence. The VSV G envelope expression plasmid hasbeen described previously (Naldini et al., (1996) In vivo gene deliveryand stable transduction of nondividing cells by a lentiviral vector.Science, 272, 263-7). HIV-1 recombinant retroviral stocks were producedusing a modification of the three-plasmid expression system described byNaldini et al., 1996. 6×10⁶ human kidney 293T cells were co-transfectedwith 10 μg packaging construct WT or integrase D64V mutant, 8 μg HIV-Luctransfer vector and 5 μg VSV G envelope protein expression plasmidsusing Lipofectamine-2000 reagent (Gibco-BRL). 48 hours post transfectionretrovirus-containing cell culture supernatants were harvested, filteredthrough 0.45 μM cellulose acetate membranes and stored at −80° C.Recombinant HIV-1 viral titres were estimated using the HIV-1 p24 gagantigen ELISA kit from Beckman-Coulter, according to the manufacturers'instructions.

[0447] Retroviral Transductions (LUCIA).

[0448] For the HIV-1 based luciferase assays (LUCIA), Jurkat T-cells(suspension cultures) were transduced with HIV-Luc recombinant viruscontaining supernants at an MOI of 0.5 in the presence of 8 μg/mlpolybrene at 37° C. for 1 hour. Cells were washed and then plated inmultiple wells (3×10⁴ cells/well) of a 96-well opaque-white tissueculture plate (Corning) containing different concentrations ofinhibitors. For HeLa cells (adherent cells) were plated and allowed toattach for 24 hours before exposure to virus containing supernatants.Cells were incubated at 37° C. for 48 hours post virus addition andLuciferase activity was quantified on a Packard TopCount-NXT microplatescintillation counter using Bright-Glo luciferase assay reagent (PromegaCorp.). The standard error (S.E.) given for all quantified transductionexperiments is calculated from at least three independent experiments.Cytotoxicity was evaluated in parallel with LUCIA (but without virus)using the commercially available CellTiter-96 AQ_(ueous) one solutioncell proliferation assay (Promega Corp.), according to themanufacturers' instructions.

[0449] HIV-1 4-day Replication Assays

[0450] C8166 T-cells were washed and infected with HIV-1 (strainsHXB2_(wt) and HXB2_(AZTres), RT amino acid changes 67N, 70R, 215F, 219Q)at low multiplicity of infection for one hour at room temperature. Thecells were then washed and distributed (5×10⁴ cells/well) in triplicateinto the wells of 96-well cell culture plates containing differentconcentrations of inhibitors. The plates were then incubated at 37° C.for 4 days. The cell-free culture fluid was then harvested and assayedfor levels of p24 viral antigen using a commercially available ELISA kit(Murex), according to the manufacturers' instructions. Cytotoxicity wasevaluated by distributing (5×10⁴ cells/well) uninfected C8166 T-cellsinto triplicate wells of 96-well cell culture plates containingdifferent concentrations of inhibitor and incubating the plates at 37°C. After 4 days, 25 μl of XTT, which is metabolised by viable but notdead cells was added and the plates incubated for a further 3 hours at37° C. Finally, the absorbance was read at a wavelength of 450 nm.

Results

[0451] In vitro ATM Assays

[0452] Compounds were assayed for ATM inhibition activity using themethod described above. Some results are detailed below in Table 1 asIC₅₀ values (the concentration at which 50% of the enzyme activity isinhibited). These are determined over a range of differentconcentrations, normally from 100 μM down to 1 nM. Such IC₅₀ values areused as comparative values to identify increased compound potencies.TABLE 1 Compound IC₅₀ - ATM 4 <200 nM 5 <200 nM 6 <2 μM 7 <200 nM 9 <20μM 13 <20 μM 17 <200 nM 18 <200 nM

[0453] The following compounds had IC₅₀ values of less than 200 nM:19-43, 44-87, 93, 102, 106-107, 109-113, 115-117, 119, 122-124, 126-131,133-135, 137-140, 142-182.

[0454] The following compounds had IC₅₀ values of less than 2 μM, inaddition to those listed above: 88-92, 94-101, 103-105, 108, 114, 118,120-121, 125, 132, 136 and 141.

[0455] Sensitisation of Cells to Ionising Radiation or DNA Double StrandBreak Chemotherapies

[0456] The data shown in FIGS. 1-4 clearly show that inhibiting ATM withcompound 4 has a significant effect on sensitising tumour derived celllines to DNA double strand break inducing agents.

[0457] Retroviral Transductions (LUCIA)

[0458] Compound 4 (known as Ku0064) was tested for its ability torepress retroviral infections using HIV-1 based LUCIA (FIG. 5).

[0459] It was found to efficiently inhibit HIV-1 LUCIA at low micromolarconcentrations in Jurkat T-cells (FIG. 5) as well as all other ATMproficient cell lines tested. The 50% inhibitory concentration (IC₅₀)for Compound 4 in LUCIA was around 1-2 μM in Jurkat cells (FIG. 5) andin the range of 1 to 10 μM for all other cell lines tested.

[0460] Compound 4 was also tested for cytotoxic and growth inhibitioneffects in parallel to LUCIA to ensure that this was not the reason forthe observed reduction in transduction efficiency. At concentrations upto 10 μM, compound 4 exposure showed no significant cytotoxic effects onJurkat cells during the course of the assay (FIG. 5).

[0461] HIV-1 based LUCIA was performed on HeLa cells in the presence ofincreasing concentrations of both compound 4 and the nucleoside analogreverse transcriptase inhibitor, 3′-azido-3′-deoxythymidine (AZT).

[0462]FIG. 7 shows that the 6ombination of compound 4 and AZT was foundto act more effectively in inhibiting HIV-1 infection than either drugalone. FIG. 7 provides an example in which increasing concentrations ofAZT was found to enhance the effectiveness of a compound of the presentinvention in inhibiting HIV-1 infections.

[0463] HIV Replication Inhibition

[0464] A replication competent HIV-1 strain (HIV_(HXB2)) was used toinfect C8166 T-cells in the absence or presence of increasingconcentrations of compound 4 (FIG. 8), in order to demonstrate theeffectiveness of compounds of the present invention in a system whereHIV replication occurs. After 4-days of virus replication, the amount ofHIV-1 in cell culture supernatants was quantified by p24 antigen ELISA.As a control, the RT inhibitor AZT was used in parallel experiments.FIG. 8A shows the inhibition of HIV-1 replication of a wild type HIV-1strain (HIV_(HXB2) wt) by compound 4 and AZT. The IC₅₀ concentration ofCompound 4 for HIV-1 replication inhibition was 0.1 μM. AZT showed anIC₅₀ of 0.002 μM.

[0465] 4-day replication assays were performed using an AZT drugresistant strain of HIV-1 (HIV_(HXB2) AZTres) in the absence or presenceof increasing concentrations of compound 4 (FIG. 8B, Table 2). The IC₅₀concentration of Compound 4 for HIV-1 replication inhibition on the AZTresistant strain was 0.06 μM. AZT showed an IC₅₀ of 0.05 μM (Table 2),thereby demonstrating a 25-fold resistance to AZT when compared to thewild type strain.

[0466] Compound 4 inhibited HIV-1 replication equally well on wild-typeHIV-1 (IC₅₀=0.1 μM; FIG. 8A, Table 2) as on the AZT resistant HIV-1strain (IC₅₀=0.06 μM; FIG. 8B, Table 2). These data provide evidencethat compounds of the present invention may be effective in both thetreatment of wild-type and acquired AZT resistant HIV-1 infections and,by implication, HIV-1 strains resistant to other drugs that target viralproteins.

[0467] Compound 4 was also tested for cytotoxic and growth inhibitioneffects on C8166 cells in parallel to the HIV-1 replication assays toensure that this was not the reason for the observed effects of viraltitre (FIG. 8C). Exposure of C8166 cells to Compound 4 showed nosignificant cytotoxic effects during the course of the assay or over theeffective concentration range (less than 1 μM) shown to inhibit HIV-1replication. The 50% cytotoxic concentration (CC₅₀) of compound 4 onC8166 cells was estimated to be greater than 20 μM. Table 2 summarisesthe experiments showing the anti-HIV-1 activity of Compound 4 in 4-dayreplication assays. Interestingly, the IC₅₀ in LUCIA (1 μM; FIG. 5) wasobserved to be 10-fold higher than in the replication assays (0.1 μM;FIG. 8A). This difference can be explained by the fact that inreplication assays multiple rounds of infection occur and each roundprovides the potential for HIV-1 inhibition. Therefore, the inhibitoryeffect of Compound 4 becomes compounded in HIV-1 replication assays. Theestimated IC₅₀ concentrations in replication assays may thereforerepresent a more accurate reflection of the extent of inhibition thatmay be seen in HIV-1 infected patients. TABLE 2 HIV-1 HXB2 (IC₅₀ μM)HIV-1 HXB2 (IC₅₀ μM) Compound Wild type AZT resistant Compound 4 0.10.06 AZT 0.002 0.05 (25 fold resistant)

1. A compound of formula I:

and isomers, salts, solvates, chemically protected forms, and prodrugsthereof, wherein: one of P and Q is O, and the other of P and Q is CH,where there is a double bond between whichever of Q and P is CH and thecarbon atom bearing the R³ group; Y is either O or S; R¹ and R² areindependently hydrogen, an optionally substituted C₁₋₇ alkyl group,C₃₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or may together form,along with the nitrogen atom to which they are attached, an optionallysubstituted heterocyclic ring having from 4 to 8 ring atoms; R³ is aphenyl or pyridyl group, attached by a first bridge group selected from—S—, —S(═O)—, —S(═O)₂—, —O—, —NR^(N)— and CR^(C1)R^(C2)— to anoptionally substituted C₅₋₂₀ carboaryl group, in which one aromatic ringatom may be replaced by a nitrogen ring atom; the phenyl or pyridylgroup and optionally substituted C₅₋₂₀ carboaryl group being optionallyfurther linked by a second bridge group, which is bound adjacent thefirst bridge group on both groups so as to form an optionallysubstituted C₅₋₇ ring fused to both the phenyl or pyridyl group and theC₅₋₂₀ carboaryl group, the phenyl or pyridyl group being furtheroptionally substituted; wherein RN is selected from hydrogen, an estergroup, an optionally substituted C₁₋₇ alkyl group, an optionallysubstituted C₃₋₂₀ heterocyclyl group and an optionally substituted C₅₋₂₀aryl group; and R^(C1) and R^(C2) are independently selected fromhydrogen, an optionally substituted C₁₋₇ alkyl group, an optionallysubstituted C₃₋₂₀ heterocyclyl group and an optionally substituted C₅₋₂₀aryl group.
 2. A compound according to claim 1, of formula Ia:


3. A compound according to claim 1, wherein Y is O.
 4. A compoundaccording to claim 1, wherein R¹ and R² form, along with the nitrogenatom to which they are attached, a heterocyclic ring having 6 ringatoms.
 5. A compound according to claim 5, wherein R¹ and R² form, alongwith the nitrogen atom to which they are attached, a group selected frommorpholino and thiomorpholino
 6. A compound according to claim 1,wherein the phenyl or pyridyl group is a phenyl group.
 7. A compoundaccording to claim 1, wherein the phenyl or pyridyl ring or the C₅₋₂₀carboaryl group in R³ bear a substituent selected from the groupconsisting of acylamido, sulfonamino, ether, ester, amido and acyl.
 8. Acompound according to claim 1, wherein R³ is selected from the followingoptionally substituted groups


9. A composition comprising a compound according to claim 1 or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or diluent.
 10. A method of treating a condition in apatient which is known to be ameliorated by the inhibition of ATMcomprising administering to said patient a therapeutically-effectiveamount of a compound according to claim
 1. 11. A method of treatingcancer in a patient comprising administering to said patient atherapeutically-effective amount of a compound according to claim 1 incombination with ionising radiation or a chemotherapeutic agent.
 12. Amethod of treating a retroviral mediated disease in a patient comprisingadministering to said patient a therapeutically-effective amount of acompound according to claim
 1. 13. A method of inhibiting ATM in vitroor in vivo, comprising contacting a cell with an effective amount of acompound according to claim 1.